CN104568886B - A kind of dark field illumination method based on total internal reflection - Google Patents

A kind of dark field illumination method based on total internal reflection Download PDF

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Publication number
CN104568886B
CN104568886B CN201510009447.XA CN201510009447A CN104568886B CN 104568886 B CN104568886 B CN 104568886B CN 201510009447 A CN201510009447 A CN 201510009447A CN 104568886 B CN104568886 B CN 104568886B
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internal reflection
total internal
dark
image
sample cell
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CN201510009447.XA
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Chinese (zh)
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CN104568886A (en
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降雨强
石浩
黄璐
靳云峰
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中国科学院遗传与发育生物学研究所
华南师范大学
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Abstract

The present invention discloses a kind of dark field illumination method based on total internal reflection, can be applied to the optical microphotograph imaging research of micro-nano granules and biological sample etc..The dark-ground illumination to sample is realized in total internal reflection of the present invention based on illumination light, signal photoimaging and detection are used as by the use of sample scattered light or fluorescence, so as to realize the observation to micro-nano-scale sample and measurement, and there is good applicability and compatibility to imaging devices such as existing microscopes.This method using total internal reflection cause illumination light in total internal reflection prism and sample cell once or repeatedly total internal reflection, formed total internal reflection dark-ground illumination, can be used for the research fields such as nano material, biological cell and light-matter interaction.

Description

A kind of dark field illumination method based on total internal reflection

Technical field

The present invention relates to the means of illumination of micro-imaging, it particularly relates to a kind of method of dark-ground illumination, particularly profit Observation to micro-nano granules in sample cell and biological sample is realized with the total internal reflection of total internal reflection prism and sample cell and is ground Study carefully, and existing microscope or self-built imaging system can be flexibly applied to extensively.

Background technology

Imaging technique based on traditional optical imagery microscope combines new Development of Laser Technology into present extensive use Fluorescence microscope, while with the development of biomolecular science and nanometer material science etc., the object of research from micro-meter scale to Nanoscale it is progressive, the technological means of higher resolution and signal to noise ratio turns into the target that iconography is constantly pursued.Nearest nanometer The nano material that noble metal granule, CNT, quantum dot etc. have special nature is widely applied to synthesis catalytic, phototube The fields such as part, nano drug-carrying and Biological imaging.The optics such as nanogold particle has special plasma absorption and scattering Characteristic, surface be easy to chemical modification, with excellent biocompatibility the features such as, be widely used in the fields such as biology, but by When nano material size is much smaller than the wavelength of visible ray, the diffraction limit of common micro-imaging is exceeded, with traditional micro- Art can not directly observe nano-scale particle imaging.Therefore dark-ground illumination technology make use of nano particle itself to scatter in terms of imaging Property stronger optical characteristics, be highly suitable for observing nanometer sized materials.

Dark-ground illumination technology applies Tyndall effect optically in principle, when illuminating bundle enters colloidal sol, In the communication process of illumination light, light is acted on particle can be deviateed originally along different directions by certain statistical law by scattering Direction, each particulate just turns into luminous point, from side it can be seen that a light beam.General dark-ground illumination micro-imaging Technology is typically used oblique illumination and the direct light light at center is blocked using annular diaphragm, therefore illuminates the condenser used The numerical aperture that numerical aperture must be greater than image-forming objective lens can just prevent illumination light from being directly entered object lens by concentrator, and Can only be from the diaphragm surrounding of concentrator and the position do not obmubed oblique fire on the sample into sample cell.Because illuminating ray is oblique fire And angle is more than the acquisition angles of object lens, it is impossible to enter object lens, thus the visual field background observed by microscope be it is dark, can only See makes sample send light because light scattering is acted on, so as to realize the details in a play not acted out on stage, but told through dialogues observation of sample.Due to having filtered out direct light, collection Simply in sample cell particle scattered light, therefore signal to noise ratio can be effectively improved in the case of dark background.The light of individual particle dissipates Penetrate in the solution, the scale ratio particle of transverse illumination beam cross section is much greater, so incidence wave can be approximated to be plane wave, again Because distance is very big between particulate, therefore scattering of the individual particle to plane wave can be regarded as.The sky of scattered light asks distribution reflection Grain attribute different sizes, material, the scattered light spatial distribution of the particle of shape of itself just has very big difference, can be by tight The electromagnetic theories of lattice is calculated, therefore can effectively extract using dark-field imaging the physical characteristic of observing samples.Apply in general to dark The sample of field illumination is that relative distribution degree is higher, the silicon in the relatively simple thin particulate samples such as biology of background structure Algae, bacterium, yeast, tissue culture cells, protozoan etc..Applicable Non-biological specimens include crystalline mineral and chemistry, colloid Particle, comprising small field trash and polymer and ceramic sheet, porosity difference, or refractive index gradient.Dark-field microscope can be with See the sample that can't see in bright field, the form and motion energy of the biological sample that is biologically checked for being unstained Power.

But with bright field illumination distinguish be dark-ground illumination effective working region it is relatively short, when condenser is fixed on When the scope of object lens is moved or moved down more than this yardstick, due to the diverging of illuminating ray, collecting efficiency can be caused to reduce With the decline of image quality.And due to condenser must use high-NA, put sample place sample when before condenser Immersion oil is usually used between lens and micro slide, light and shade switching needs to change condenser or diaphragm, typically can not be in experiment In be switched fast, and because the high-NA of illumination also limit the thickness of sample.So sample and slide Thickness receive considerable restraint, therefore traditional dark-ground illumination sample receives very big limitation.

Another utilization total internal reflection reaches that the technology of nanometer resolution is total internal reflection micro-imaging technique, passes through profit The characteristic of evanescent wave is produced after being totally reflected with light in medium another side, excites fluorescence molecule to observe the pole of fluorescence calibration sample Thin region, the dynamic range of observation is typically not greater than in 200nm or so.Because by exciting for total internal reflection back reflection face side Light is exponentially decayed, and only the sample areas extremely close to fully reflecting surface can produce fluorescent reflection, largely reduce background Optical noise disturbance-observer target, can help researcher to obtain high-quality image quality and reliable observation data.But sample Thickness and areas imaging still receive very big limitation.

The purpose of the present invention is to overcome existing details in a play not acted out on stage, but told through dialogues and total internal reflection evanescent field illumination technology in thickness of sample and imaging There is provided a kind of lower cost for not enough in terms of scope, it is easy to accomplish utilization illumination light and sample effect scattering properties into The new dark field illumination method of picture.This method can realize above and below both sides while the function such as observe and excite, it is simple in construction can be with Existing imaging, detection and activating system are combined, and it is convenient to adjust, it is possible to achieve the illumination to micro-nano granules in thick sample, and then Easily facilitate the dynamically research such as measurement of development associated biomolecule image accurate surveying and colloid property.

The content of the invention

The purpose of the present invention be overcome existing details in a play not acted out on stage, but told through dialogues and total internal reflection illumination technology in terms of thick sample sizes not There is provided the method that can be used for micro-nano granules to observe and study that a kind of scattering properties of utilization illumination light is imaged for foot.This method Both sides are observed and arousal function simultaneously above and below can realizing, simple in construction to be combined with existing imaging, detection and activating system, are adjusted Section is convenient, it is possible to achieve the illumination to thick sample, and then easily facilitates the observation of development associated biomolecule image and colloid property etc. and grind Study carefully.

For up to above-mentioned purpose, a kind of dark-ground illumination device based on total internal reflection of the invention, using following technical side Case:

A kind of dark-ground illumination device based on total internal reflection, including total internal reflection dark-ground illumination microscope, total internal reflection are dark Illumination microscope in field includes objective table, upper image-forming objective lens, total internal reflection prism, lower image-forming objective lens, and total internal reflection dark-ground illumination shows The objective table of micro mirror is provided with sample cell, and sample cell, which is provided with total internal reflection prism, sample cell, loads sample gold nano grain The aqueous solution, total internal reflection dark-ground illumination microscope is provided with symmetrically upper light path and lower light path, and total internal reflection dark-ground illumination is micro- The upper light path side of mirror is provided with upper image-forming objective lens, and the microscopical lower light path side of total internal reflection dark-ground illumination is provided with lower imaging thing Mirror, the imaging surface of the microscopical upper light path of total internal reflection dark-ground illumination and lower light path overlaps each other the area to be seen in sample cell In domain;

Imaging photosensor on the microscopical upper optical routing of total internal reflection dark-ground illumination, imaging len B, optical filter A, on Image-forming objective lens are constituted, and upper image-forming objective lens are provided with the upside of total internal reflection dark-ground illumination microscope, upper image-forming objective lens are away from total internal reflection Imaging len B is provided with before the microscopical opposite side of dark-ground illumination, imaging len B is set before the opposite side away from upper image-forming objective lens Have and upper imaging photosensor is provided with before optical filter A, opposite sides of the optical filter A away from imaging len B, total internal reflection details in a play not acted out on stage, but told through dialogues is shone The light beam that bright microscopical upper light path is sent is irradiated to imaging photoelectric transfer by upper image-forming objective lens, imaging len B, optical filter A Sensor;

(lower light path) is provided with lower image-forming objective lens on the downside of total internal reflection dark-ground illumination microscope, and lower image-forming objective lens are away from complete interior anti- Penetrate the microscopical opposite side of dark-ground illumination provided with one with light path into 45 degree of angles dichroscope, dichroscope sideways and lower imaging Object lens light path vertical direction is provided with laser, and beam-expanding collimation lens group, the light of laser are provided between laser and dichroscope Road is reflexed to up to lower image-forming objective lens after beam-expanding collimation lens group by dichroscope, focuses on sample cell.

Further, in certain embodiments, the dark-ground illumination device of the total internal reflection also comprising laser illuminator, it is complete in it is anti- Penetrate dark-ground illumination microscope, total internal reflection prism, sample cell.

Further, in certain embodiments, speculum normal thereto, mirrored side are provided with below the dichroscope Have an imaging len A and optical filter B with lower image-forming objective lens light path vertical direction, the imaging beams of lower image-forming objective lens by two to After Look mirror, speculum, imaging len A and optical filter B, the image of amplification is projected on lower imaging photosensor.

Further, in certain embodiments, the laser that the laser illuminator is sent passes through total internal reflection prism total internal reflection In the aqueous solution for the micro-nano granules being irradiated in sample cell, laser illuminator enters again after the total internal reflection by sample cell bottom of pond Enter total internal reflection prism, prism is left in multiple reflections outgoing in total internal reflection prism.

Further, in certain embodiments, the microscopical excitation light path of total internal reflection dark-ground illumination is by laser, expansion Beam collimation lens set, dichroscope, lower image-forming objective lens composition, total internal reflection dark-ground illumination microscope are another away from lower image-forming objective lens Side (upper light path) is provided with upper image-forming objective lens;The dichroscope that lower image-forming objective lens are placed provided with 45 degree of angle first below, two It is beam-expanding collimation lens group, laser to Look mirror deflection laser side, laser sends laser by beam-expanding collimation lens group, arteries and veins Develop Shu Houjing and cross dichroscope and reflected and deflect 90 ° of lower image-forming objective lens of entrance, converge in sample cell.

Further, in certain embodiments, sample cell slide is provided with the sample cell, sample cell bottom is provided with 0.12 Sample cell cover glass thick~0.2mm.

Further, in certain embodiments, the lower image-forming objective lens are:The micro objective of 20x, 50x, 100x equimultiple, Upper image-forming objective lens are:The micro objective of 20x, 50x, 100x equimultiple;640~1060nm of optical maser wavelength of laser is adjustable, band When wide 10nm, the pulsewidth of femtosecond is about 100fs, and the power of light extraction is about 1000mW, is into microscopical power 800mW;Upper image-forming objective lens, lower image-forming objective lens:The NA of object lens is 0.65, and operating distance is 4.5mm, can continuously calibrate 0 and arrive Cover-glass thickness or silicon wafer thickness thick 1.2mm.

A kind of dark field illumination method based on total internal reflection, exemplary method is as follows:

It is provided with sample cell in total internal reflection prism, side illumination laser angle and position can be to meet total internal reflection critical Adjusted in angular range, the module of wherein fluorescence excitation is replaced the binomial Look mirror of matching exciting light, laser is imported for reflecting The femtosecond light beam and transmission illumination light or fluorescence of device, upper light path device build imaging system;

The observing samples of sample cell can be micro-nano granules, by the gold of the 60nm diameters of 4 DEG C of Cord bloods before experiment Nanoparticles solution, with 100~2000 times of deionized water or distilled water diluting, is then used after 5~10min of ultrasound, is made Grain is uniformly distributed in the solution;

The gold nano grain solution configured is placed in the sample cell being cleaned by ultrasonic, covered with clean prism close On envelope, the center for being placed into the microscopical objective table of total internal reflection dark-ground illumination, under the conditions of light field, sample cell is found Bottom, then opens lower imaging photosensor, upper imaging photosensor, image is shown on computers, is easy to regulation;

Scattered light or fluorescence that the fluorescence and laser illuminator of laser excitation sample interact with sample, by sample cell one Part enters upper image-forming objective lens, is partly into lower image-forming objective lens, remaining partly dissipative falls;

Fluorescence or scattered light into lower image-forming objective lens pass through dichroscope, then be reflected by a reflector and deflect by into As lens A and optical filter B, gathered by lower imaging photosensor;

Fluorescence or scattered light into upper image-forming objective lens pass through imaging len B and optical filter A, by upper imaging photoelectric sensing Device is gathered;

Laser is opened, by adjusting speculum, the angles and positions of incident laser are adjusted so that laser is after prism Experiences total internal reflection;Fine adjustment laser and sample cell so that laser has just incided the bottom at sample cell center, in display screen It is observed that correct position is arrived in regulation.It is observed that visual field background is relatively low in place, mainly non-imaged face The scattered light of particle and sample cell etc., then adjust object lens position, it can be seen that the bright scattered light of particle.Finally adjustment laser is strong The parameter of degree and photoelectric sensor, obtains suitable image;

Under the conditions of details in a play not acted out on stage, but told through dialogues total internal reflection illumination, it is observed that the Brownian movement of particle in the solution, then opens and swashs Light device;Two-photon fluorescence can be carried out to excite or the micro- manipulation research of optics.

Present invention can apply to the total internal reflection of the imaging of the optical microphotograph of micro-nano granules and biological sample etc. and research is dark Field means of illumination.The dark-ground illumination to sample is realized in total internal reflection based on illumination light, is made using sample scattered light or fluorescence For signal photoimaging and detection, it is possible to achieve observation and measurement to micro-nano-scale sample, and to existing illumination and imaging Device has good applicability and compatibility.The core of this method is using total internal reflection so that illumination light is in total internal reflection Multiple total internal reflection in prism and sample cell, forms total internal reflection dark-ground illumination, it is possible to achieve to the photograph of micro-nano-scale sample The functions such as bright and imaging.

Brief description of the drawings

Fig. 1 show the structural representation of the embodiment of the present invention;

Fig. 2 show the total internal reflection Dove prism and sample pool example of the embodiment of the present invention;

Fig. 3 show the integrated processing instance of total internal reflection Dove prism and sample cell of the embodiment of the present invention;

Fig. 4 show the integrated processing instance of total internal reflection arc prism and sample cell of the embodiment of the present invention;

Fig. 5 show the example of the total internal reflection dark-ground illumination imaging of the embodiment of the present invention.

Description of reference numerals is as follows:

Laser 101, lower imaging photosensor 102, upper imaging photosensor 103, dichroscope 104, speculum 105th, beam-expanding collimation lens group 201, imaging len A 202, imaging len B 203, total internal reflection dark-ground illumination microscope 300, Upper image-forming objective lens 301, total internal reflection prism 302, laser illuminator 303, sample cell 304, lower image-forming objective lens 305, sample cell carry glass Piece 311, sample cell cover glass 312, optical filter A 401, optical filter B 402.

Embodiment

For the feature, technological means and the specific purposes reached, function of the present invention can be further appreciated that, this hair is parsed Bright advantage and spirit, are further understood by below in conjunction with accompanying drawing with embodiment to the detailed description of the present invention.

For the feature, technological means and the specific purposes reached, function of the present invention can be further appreciated that, this hair is parsed Bright advantage and spirit, are further understood by below in conjunction with accompanying drawing with embodiment to the detailed description of the present invention.

The present invention includes total internal reflection dark-ground illumination microscope 300, and referring to shown in accompanying drawing 1, total internal reflection dark-ground illumination shows Sample cell 304 is placed on the objective table of micro mirror 300, sample cell 304 is provided with total internal reflection prism 302, and sample cell 304 is inner to be loaded The aqueous solution of sample gold nano grain, total internal reflection dark-ground illumination microscope 300 is provided with symmetrically upper light path and lower light path, complete interior The upper light path side for reflecting dark-ground illumination microscope 300 is provided with upper image-forming objective lens 301, total internal reflection dark-ground illumination microscope 300 Lower light path side be provided with lower image-forming objective lens 305, the imaging of the upper light path of total internal reflection dark-ground illumination microscope 300 and lower light path Face overlaps each other in the aqueous solution of the gold nano grain of sample cell 304.

Total internal reflection dark-ground illumination microscope 300 includes objective table, lower image-forming objective lens 305, upper image-forming objective lens 301.

Lower image-forming objective lens 305 are:50x micro objective, upper image-forming objective lens 301 are:50x micro objective.

Imaging photosensor 103 on the upper optical routing of total internal reflection dark-ground illumination microscope 300, imaging len B 203, Optical filter A 401, upper image-forming objective lens 301 are constituted, and the opposite side of total internal reflection dark-ground illumination microscope 300 is provided with lower image-forming objective lens 305, opposite side of the lower image-forming objective lens 305 away from total internal reflection dark-ground illumination microscope 300 passes through dichroscope 104 and speculum There are imaging len A 202 and optical filter B 402 after 105, imaging beam will after imaging len A 202 and optical filter B 402 The image of amplification is projected on lower imaging photosensor 102.Total internal reflection dark-ground illumination device mainly have laser illuminator 303, Total internal reflection dark-ground illumination microscope 300, sample cell 304 are constituted.

The laser that laser illuminator 303 is sent is irradiated to the gold in sample cell 304 by the total internal reflection of total internal reflection prism 302 In the aqueous solution of nano particle, laser illuminator 303 is again introduced into total internal reflection rib after the total internal reflection by the bottom of pond of sample cell 304 Mirror 302, prism is left in multiple reflections outgoing in total internal reflection prism 302.

The excitation light path of total internal reflection dark-ground illumination microscope 300 from laser 101, beam-expanding collimation lens group 201, two to Look mirror 104, lower image-forming objective lens 305 are constituted, opposite side of the total internal reflection dark-ground illumination microscope 300 away from lower image-forming objective lens 305 (upper light path) is provided with upper image-forming objective lens 301.The lower downside of image-forming objective lens 305 is first provided with the dichroscope that 45 degree of angle is placed 104, the deflection laser of dichroscope 104 side is laser 101, and beam-expanding collimation lens group 201, laser 101 sends laser warp Beam-expanding collimation lens group 201 is crossed, pulse is reflected by dichroscope 104 after expanding and deflects 90 ° of lower image-forming objective lens of entrance 305, converge in sample cell 304.

The scattered light or fluorescence that the fluorescence and laser illuminator 303 of laser excitation sample interact with sample are by sample cell 304 are partly into image-forming objective lens 301, are partly into lower image-forming objective lens 305, and remaining partly dissipative falls.Into it is lower into As the fluorescence or scattered light of object lens 305 pass through dichroscope 104, reflected and deflected by imaging len A by speculum 105 202 and optical filter B 402, is gathered by lower imaging photosensor 102.Into the fluorescence or scattered light of upper image-forming objective lens 301 By imaging len B 203 and optical filter A 401, gathered by imaging photosensor 103.

Fig. 2 is total internal reflection Dove prism and sample cell part separates example, and total internal reflection prism 302 is below sample cell Slide 311, sample cell slide 311 is below sample cell cover glass 312.

Fig. 3 is total internal reflection Dove prism and the integrated processing instance of sample cell, and sample cell 304 (trapezoidal) is arranged in complete In reflecting prism 302, sample cell cover glass 312 is arranged below in sample cell 304.

Fig. 4 is total internal reflection arc prism and the integrated processing instance of sample cell, the sample cell with total internal reflection prism 302 Sample cell cover glass 312 is arranged below in 304 (arcs), sample cell 304.

In one of the embodiments, the imaging function module of the inverted fluorescence microscope based on routine or designed, designed With arousal function module;It is positioned on the two-dimentional objective table of conventional inverted fluorescence microscope by laser illuminator, total internal reflection prism 302nd, the illumination functions module that sample cell 304 is constituted;Upper imaging optical path extendable functions module.

The imaging of system and the configuration of arousal function module are the devices in order to realize correlation function and must configure, main Will be by the lower image-forming objective lens 305 of observation, dichroscope 104, speculum 105 (high reflectance eyeglass), (imagings of imaging len A 202 Achromatic lens), optical filter B 402 (bandpass filter) and lower imaging photosensor 102 (band imaging CCD or CMOS phases Machine) composition imaging optical path;And may include continuous or pulse laser 101 what dichroscope 104 was separated, and expand The compositions such as collimation lens set 201 (extender lens group) cause system possess capture or excitation etc. function arousal function mould Block.

Illumination functions module mainly includes laser and attached modulation module as the core of invention, including it is continuous or Pulse laser, the lens-pinhole object lens of model-based optimization laser filtering, light path displacement angle control speculum group and absorb Penetrate the absorption cell or nigrescence baffle plate of laser;The importing laser being processed into by high-index material, and laser illuminator 303 is passed through Cross the total internal reflection prism 302 being repeatedly totally internally reflected back toward up to the center of sample cell 304;Sample cell 304 mainly carries total internal reflection Prism 302 simultaneously places sample, and it, which is gone to the bottom, is provided with by sample cell cover glass 312 thick 0.17mm.

Upper imaging optical path extendable functions module is by the upper image-forming objective lens 301 of another observation, (imagings of imaging len B 203 Achromatic lens), optical filter A 401 (bandpass filter) and upper imaging photosensor 103 (band imaging CCD or CMOS phases Machine) composition imaging optical path.Its set mainly depending on demand, it is therefore an objective to increase a dimension observation system, its configure with The imaging of system and arousal function module are optically and indifference.

In certain embodiments, the microscopical main optical path of total internal reflection dark-ground illumination is imaging device, and collection is complete interior anti- The scattered light of sample in dark-ground illumination is penetrated, optical filter B 402 is multi-coated interference film or optical glass, filters out exciting light, is gathered Scattered light and fluorescence.Dichroscope of the inverse beam direction of light path provided with 45 degree of angle under total internal reflection dark-ground illumination microscope 104, dichroscope 104 is higher than 95% in 750nm to 1050nm reflectivity, is more than 5%, multilayer in 350nm to 680nm transmissivities Coating designs introduce low dispersion.Total internal reflection dark-ground illumination 300 times light paths of microscope are from sample cell 304 transmitted through coming and pass through The light beam that lower image-forming objective lens 305 (focusing objective len) come out passes through dichroscope 104, speculum 105, imaging len A 202, optical filtering Lower imaging photosensor 102 is reached after piece B 402, and completes real-time in lower imaging photosensor 102 (imaging device) Imaging.The optical maser wavelength 640-1060nm of the laser 101 used is adjustable, when bandwidth 10nm, and the pulsewidth of femtosecond is about 100fs, the power of light extraction is about 1000mW, is 800mW into microscopical power.

Image-forming objective lens 301, lower image-forming objective lens 305 in observation:The NA of object lens is 0.65, and operating distance is 4.5mm, Ke Yilian Cover-glass thickness or silicon wafer thickness of the continuous calibration 0 to 2mm thickness.

The sample used is the gold nanosphere of 60 nanometers of the coated diameter of cetab.The transmission of sample Electron microscope, gold grain shape is regular, and size is substantially uniform, and size slightly has difference, therefore can be used as what is be imaged and study Object.Correspondence is because the crest of 60nm particle resonance scatterings laser spectrum is in 532nm or so, continuous green 532nm lasers, power 200mW, optical power stability fluctuation is less than 3%, and facular model TEM00, the M factor are less than 1.3.

Total internal reflection prism 302 can use Amici prism or right angle angle rib in the case of without using upper imaging optical path Mirror, light beam entrance prism enters sample cell 304 and after bottom of pond experiences total internal reflection again by prism outgoing, the profile of prism and Size can make corresponding modification.

Total internal reflection prism 302 and sample cell 304 integrated can be processed, and the bottom of sample cell 304 utilizes sample cell lid glass Piece 312 is sealed, and proprietary design reduces interface loss.

The material of total internal reflection prism 302 should apply to the comparatively ideal generation total internal reflection refractive index of energy and should be greater than solution, By taking water as an example 1.33, the optical loss of illumination light wavelength is low to be less than 1dB/km, it is easy to be processed as selection standard, what can be enumerated has The conventional optical glass such as quartz glass, K9 glass, methacryl resin, polystyrene resin, polycarbonate resin, cycloolefin tree The optical plastics such as fat, metering system-styrene copolymer resin, cycloolefin-olefine copolymer resin, polyester resin, it is every The material that conditions above can be met all can be as the raw material for processing total internal reflection prism 302.

Laser illuminator 303 can be continuous light, pulsed light;The wave-length coverage of laser includes ultraviolet light, it is seen that light, infrared light Deng the selection of wavelength is relevant with illuminated sample, while should be also consistent with the compatibility of detector and system.

A kind of dark field illumination method based on total internal reflection, specific method is as follows:

The figure experimental system such as of building of system is to be based on a kind of inverted fluorescence microscope, sample cell 304 and total internal reflection rib Placed above and below such as figure of mirror 302, side illumination laser angle and position can be adjusted meeting in the range of critical angle for total internal reflection degree, The module of wherein fluorescence excitation is replaced the binomial Look mirror of matching exciting light, and femtosecond light beam and transmission illumination light are imported for reflecting Or fluorescence, upper light path device be built into as desired as etc. system.Observing samples are gold nano grain, by 4 DEG C before experiment 100~2000 times of the gold nano grain solution deionized water or distilled water diluting of the 60nm diameters of Cord blood, Ran Houchao Used after 5~10min of sound.

The gold nano grain solution configured is placed in the sample cell being cleaned by ultrasonic, covered with clean prism close On envelope, the center for being placed into microscopical objective table.Under the conditions of light field, the bottom of sample cell is found, light is then opened Electric transducer shows image on computers, is easy to regulation.

Laser is opened, by the angles and positions for adjusting reflector alignment incident laser so that laser is after prism Experiences total internal reflection.Fine adjustment laser and sample cell so that laser has just incided the bottom at sample cell center, in display screen It is observed that correct position is arrived in regulation.It is observed that visual field background is relatively low in place, mainly non-imaged face The scattered light of particle and sample cell etc., then adjust object lens position, it can be seen that the bright scattered light of particle.Finally adjustment laser is strong Parameter of degree and photoelectric sensor etc., obtains suitable such as Fig. 5 image, can clearly tell in liquid above and below focal plane Nano particle.

Under the conditions of details in a play not acted out on stage, but told through dialogues total internal reflection illumination, it is observed that the Brownian movement of particle in the solution, then opens and swashs Light device, can further carry out two-photon fluorescence excite or optics it is micro- manipulation etc. research.

The observation to the thicker sample such as micro-nano granules in sample cell and biological sample can be realized using this invention and is ground Study carefully, can with existing imaging, detect and the system such as excite flexibly to be combined, be easy to regulation to transform, while ensure that good applicability And compatibility, it can apply to the Related Research Domains such as Biological imaging observation and colloid property.

Embodiment described above only expresses some embodiments of the present invention, and it describes more specific and detailed, but simultaneously Therefore limitation of the scope of the invention can not be interpreted as.It should be pointed out that for the person of ordinary skill of the art, Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection model of the present invention Enclose.Therefore, protection scope of the present invention should be determined by the appended claims.

Claims (4)

1. a kind of dark-ground illumination device based on total internal reflection, including total internal reflection dark-ground illumination microscope (300), laser illuminator (303), upper light path, lower light path;
Wherein total internal reflection dark-ground illumination microscope (300) includes upper image-forming objective lens (301), objective table, lower image-forming objective lens (305);
Sample cell (304) is provided with objective table, sample cell (304) is provided with total internal reflection prism (302);Laser illuminator (303) laser sent is irradiated to the micro-nano granules in sample cell (304) by total internal reflection prism (302) total internal reflection In the aqueous solution, total internal reflection prism (302), total internal reflection rib are again introduced into after the total internal reflection by sample cell (304) bottom of pond Mirror (302) uses total internal reflection Dove prism, realizes that prism is left in multiple reflections and outgoing in total internal reflection prism (302);
Upper optical routing is set gradually in opposite side of the upper image-forming objective lens (301) away from total internal reflection dark-ground illumination microscope (300) Imaging len B (203), optical filter A (401), upper imaging photosensor (103) combine, total internal reflection dark-ground illumination The light beam that microscope (300) is sent upwards is irradiated to imaging photoelectric sensing by imaging len B (203), optical filter A (401) Device (103);
Lower light path includes dichroscope (104), laser (101), beam-expanding collimation lens group (201), wherein dichroscope (104) Be arranged at lower image-forming objective lens (305) away from total internal reflection dark-ground illumination microscope (300) opposite side, and with lower image-forming objective lens (305) light path is into 45 degree of angles;Laser (101) be arranged at dichroscope (104) side, and with lower image-forming objective lens (305) light path At vertical position;Beam-expanding collimation lens group (201) is arranged between laser (101) and dichroscope (104), laser (101) light path is reflexed to up to lower image-forming objective lens (305) after beam-expanding collimation lens group (201) by dichroscope (104), Focus on sample cell (304).
2. a kind of dark-ground illumination device based on total internal reflection according to claim 1, it is characterised in that the dichroic Speculum (105) normal thereto is provided with below mirror (104), speculum (105) is vertical with lower image-forming objective lens (305) light path sideways Direction is provided with imaging len A (202) and optical filter B (402), and the imaging beam of lower image-forming objective lens (305) passes through dichroscope (104), after speculum (105), imaging len A (202) and optical filter B (402), the image of amplification is projected into lower imaging photoelectricity On sensor (102).
3. a kind of dark-ground illumination device based on total internal reflection according to claim 1, it is characterised in that the sample cell (304) sample cell slide (311) is provided with, sample cell (304) bottom is provided with sample cell cover glass thick 0.12~0.2mm (312)。
4. a kind of dark-ground illumination device based on total internal reflection according to claim 1, it is characterised in that the lower imaging Object lens (305) are:20x, 50x, 100x times of micro objective, upper image-forming objective lens (301) are:20x, 50x, 100x times micro- Endoscope objective lenses.
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