CN207163909U - A kind of living cells based on terahertz time-domain decay total reflection spectrum monitors experimental provision in real time - Google Patents
A kind of living cells based on terahertz time-domain decay total reflection spectrum monitors experimental provision in real time Download PDFInfo
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Abstract
The utility model discloses a kind of living cells based on terahertz time-domain decay total reflection spectrum to monitor experimental provision in real time, the characteristics of penetration depth for being totally reflected evanescent wave decay using Terahertz with cellular layer thickness matching, terahertz time-domain decay total reflection measurement is carried out using double ATR prisms, time domain electric field and complex dielectric constant of the cell in terahertz wave band are directly obtained by terahertz time-domain spectroscopic technology（Or complex index）The real-time monitoring to whole cell interior physiological change process can be achieved, avoid traditional transmission-type and limitation that reflective tera-hertz spectra is easily influenceed by polar liquid high-absorbility, it also avoid the shortcomings that conventional cell biological method is needed to cell dyeing and intrusion；The utility model optical system integrated level is high, ensures the accuracy of Terahertz light path, eliminates the regulation of optical element in experiment, it is convenient to realizes the portable of whole spectroscopic system and movement, is well suited for using in Cell Biology Experiment room.
Description
Technical field
The invention belongs to terahertz light spectral technology and application field, and in particular to one kind is all-trans based on terahertz time-domain decay
Penetrate spectrum（THz TD-ATR spectrum）Measurement and analytical equipment, the present invention be applied to the life such as external living cells physiology and pathology
The real-time monitoring of process.
Background technology
Cell is the base unit of life entity structure and vital movement.Growing for life entity is bred, carefully by cell
Born of the same parents' differentiation realized with Apoptosis, and the pathogenesis of all diseases is also based on cytopathy is studied.Traditional cell life
Thing method includes protein immunization imprinting（Western Blot）, enzyme linked immunological group（ELISA）With cellular immunity group（IC）
Deng, it is necessary to which the sample pre-treatments of complexity, such as cell is fixed, constituents extraction, fluorescence labeling.Invasive, consumption be present in these technologies
When and the shortcomings of false positive/negative findings, it is difficult to obtain the specifying information and prison of intracellular matter structure in real time, in situ
Survey physiology or the pathologic processes such as cell propagation, differentiation, apoptosis, necrosis.
At present, physiology or pathologic process based on spectral technique monitoring living cells mainly include fluorescence spectrum, infrared spectrum,
Laser Raman spectroscopy.Fluorescence spectrum depends on fluorescence probe, and the introducing of fluorescence probe may cause to the physiological status of cell
It is certain to influence；Infrared spectrum is larger by water ambient interferences；The raman scattering spectrum of water is extremely weak, and the Raman of other biological material is believed
Breath is but very abundant, therefore Raman spectrum is suitable to research measure living cells especially continuous monitoring single living cell, but Raman noise
Than not high, it is difficult to therefrom obtain the obvious spectrum cutting edge of a knife or a sword about material molecule, generally use improves laser power, increase time for exposure etc.
Method improves signal to noise ratio, but to increase cell disturbed so that impaired risk for these methods.The vibration and rotation of large biological molecule
Energy level is in terahertz wave band more, while THz wave photon energy is low, will not cause ionising radiation, suitable for biomolecule,
The diagnosis detection research of cell and tissue.But traditional terahertz light spectral technology is also vulnerable to the influence of water environment, therefore simultaneously uncomfortable
In the in-situ monitoring of living cells.
The content of the invention
The purpose of the present invention is the physical characteristic based on terahertz light, is proposed a kind of based on terahertz time-domain decay total reflection
Spectrum（THz TD-ATR spectrum）Analysis and measurement apparatus, with solve the life processes such as external living cells physiology and pathology are entered
The problem of row monitoring in real time.
In order to meet above-mentioned above-mentioned purpose, the present invention uses following technological means：
A kind of living cells based on terahertz time-domain decay total reflection spectrum monitors experimental method, including following step in real time
Suddenly：
Step 1：It is seeded in after living cells digestion is resuspended on the ATR silicon prisms that sterilization is crossed, in cell culture fluid
Culture, makes living cells in the superficial growth close to ATR silicon prisms；
Step 2：During measurement, first set using an ATR silicon prism without living cells as with reference to ATR prisms to too
In hertz light path, its terahertz time-domain electric field is measured；
Step 3：Set into Terahertz light path, measure using the ATR silicon prism with living cells as sample ATR prisms
Its terahertz time-domain electric field；
Step 4：FFT is carried out using the reference light and sample light Terahertz electric field of acquisition, it is each to obtain its
From complex frequency domain electric field, obtain system complex transfer function than reference light frequency domain electric field with sample optical frequency domain electric field, in conjunction with
Plural Fresnel equation is solved, and obtains the complex dielectric constant of cell sample；
Step 5：In repeat step one to four at different moments, the change of sample complex dielectric constant is obtained, and combine micro-
Mirror observed result, the physiological change process of cell sample is monitored.
In the above-mentioned technical solutions, the concentration after a kind of living cells digestion is resuspended is 1x106 cells/mL。
In the above-mentioned technical solutions, the living cells is 37 °C in temperature, contains 5% CO2Cultivated in cell culture fluid.
A kind of living cells based on terahertz time-domain decay total reflection spectrum monitors experimental provision in real time, including：
The mechanical platform for positioning and clamping for optical element, is provided with guide rail on the mechanical platform, on the guide rail
It is provided with the sample ATR silicon prism that can be slided along guide rail and refers to ATR prisms, the sightingpiston of the sample ATR silicon prisms
On be provided with culturing room,
The guide rail both sides are respectively arranged with one group of off axis paraboloidal mirror, and the off axis paraboloidal mirror of side is used for Terahertz
The light of emitting head is pooled to the ATR prism regions on guide rail, and the off axis paraboloidal mirror of opposite side is used to terahertz light being pooled to
Terahertz is popped one's head in,
Reflecting microscope is provided with above Terahertz light path on the guide rail, for real-time monitored sample ATR prisms
On living cells change,
The sample ATR silicon prism, with reference to ATR prisms pass through optical fiber or cable connection to system host.
In the above-mentioned technical solutions, shown sample ATR silicon prism and reference ATR prisms are based on the complete of High Resistivity Si making
Reflecting prism, the refractive index of the prism is 3.42, and resistivity is more than 2 k Ω .cm.
In the above-mentioned technical solutions, the culturing room on the sample ATR silicon prisms is pasted onto sample ATR silicon ribs including one
The Fence structure on mirror surface, the Fence structure form the seal cavity of a upper end open with sample ATR silicon prism surface.
In the above-mentioned technical solutions, the opening of the seal cavity is provided with the quartz window that can be picked and placeed, the stone
English window is transparent window.
In the above-mentioned technical solutions, one group of off axis paraboloidal mirror includes two 90 ° of off axis paraboloidal mirrors, two groups four
90 ° of off axis paraboloidal mirrors and Terahertz probe, terahertz sources head form the decay total reflection measurement of 8f integrated forms terahertz time-domain
Optical system.
In the above-mentioned technical solutions, the guide rail is a V-groove being made by metal material, and the V-groove is used
In support sample ATR prisms and refer to ATR prisms.
In the above-mentioned technical solutions, the sample ATR prisms and bottom with reference to ATR prisms are V-type shape, the V with guide rail
Mutual of type groove.
In the above-mentioned technical solutions, the left and right sides is respectively arranged with a window on shown guide rail, for passing through for light path.
In summary, by adopting the above-described technical solution, the beneficial effects of the invention are as follows：
1. realizing the tera-hertz spectra response measurement of living cells using terahertz time-domain Technique of Attenuated Total Reflectance, its advantage exists
In being totally reflected the characteristics of penetration depth of evanescent wave is with cellular layer thickness matching using Terahertz decay, pass through terahertz time-domain light
Spectral technology directly obtains time domain electric field and complex dielectric constant of the cell in terahertz wave band（Or complex index）, achievable pair
The real-time monitoring of whole cell interior physiological change process, avoids traditional transmission-type and reflective tera-hertz spectra easily by polarity
The limitation that liquid high-absorbility influences, it also avoid the shortcomings that conventional cell biological method is needed to cell dyeing and intrusion.
2. make decay total reflection using High Resistivity Si（ATR）Prism, it the advantage is that using High Resistivity Si in terahertz wave band
The characteristics of high index of refraction, low absorption, it can be achieved that there is larger ranges of indices of refraction sample（Such as cell and nutrient solution）Decay be all-trans
Measurement is penetrated, and the transmission loss of THz wave in the prism can be reduced.
3. making cell culture chamber using the method that " fence " is directly pasted in ATR prisms upper surface, realize that work to be measured is thin
Born of the same parents the advantage is that the living cells layer using adherent growth in the adherent growth of silicon prism surface, can make to pass along silicon prism surface
Defeated Terahertz evanescent wave only fully acts on tested cell, the influence of environmental factor is reduced, so as to obtain the degree of accuracy more
High cell tera-hertz spectra response data.
4. carrying out terahertz time-domain decay total reflection measurement using double ATR prisms, joined with one of them empty ATR prism
Photo measure is examined, another ATR prism equipped with sample carries out sample photo measure, and sliding realization with guide rail refers to ATR prisms and sample
Measures conversion between product ATR prisms, the cell sample terahertz at each moment is obtained by alternately measurement reference light and sample light
Hereby spectrum, it the advantage is that the alternating for carrying out reference light and sample light using being slided in short time interval using guide rail is measured,
The ATR prism alignment errors that experimental implementation can be avoided to introduce, and terahertz light source signal itself fluctuation can be reduced to measurement result
Influence, overcome when being measured only with an ATR prism, lacking with reference to photo measure can not be carried out at each moment
Point.
5. cell morphology is become using setting the mode of reflecting microscope to realize above the cell culture chamber of ATR prisms
The real-time monitored of change, it the advantage is that and obtain cell physiological state using microscope real-time monitored, it is convenient to realize and real-time
The direct comparison of tera-hertz spectra, decay so as to more accurately obtain cell physiological state change at different moments with cell Terahertz
The corresponding relation of total reflection spectrum change.
6. by the use of 4 90 ° of off axis paraboloidal mirrors as thz beam transmission and collimation optics, binding modulesization are too
Hertz transmitting and detection photoconductive antenna form the decay of 8f integrated forms terahertz time-domain and are totally reflected measurement optical system, and its advantage exists
In using integrated optical system framework, add based on the mechanical grip mode for rationally designing optical element and using rational machinery
Work precision ensures the accuracy of Terahertz light path, eliminates the regulation of optical element in experiment, it is convenient to realize whole light
The portable and movement of spectra system, is well suited for using in Cell Biology Experiment room.
Brief description of the drawings
Examples of the present invention will be described by way of reference to the accompanying drawings, wherein：
Fig. 1 is the terahertz time-domain decay total reflection spectrum experimental system schematic layout pattern monitored in real time for living cells；
Fig. 2 is sample ATR prisms and reflecting microscope schematic layout pattern with cell culture chamber；
Wherein：01 is terahertz sources head;02 is terahertz detection head；03~06 is 90 ° of off axis paraboloidal mirrors；07 is
With reference to ATR prisms；08 is sample ATR prisms（With cell culture chamber）, 8-2 is cell culture chamber, and 8-3 is adherent layer,
8-4 is cell culture fluid；09 is reflecting microscope；10 be guide rail（Slided for ATR prisms）；11 and 12 be for optics member
Part positions and the mechanical structure of clamping；13 be all -fiber terahertz time-domain spectroscopy system host；14th, 15,16 be respectively optical fiber and
Cable, 17 be computer.
Embodiment
All features disclosed in this specification, or disclosed all methods or during the step of, except mutually exclusive
Feature and/or step beyond, can combine in any way.
As shown in Fig. 1, terahertz sources head is swashed by polarization maintaining fiber pigtail and cable in additional femtosecond in this programme
The pumping of light pulse and the lower psec terahertz pulse that produces of bias generator loading radiate；Terahertz probe by polarization maintaining fiber pigtail and
Cable, using additional femto-second laser pulse pumping, and lock-in amplifier detection and fibre delay line scanning are combined, realized to skin
The time domain electric field waveform detection of second terahertz pulse；Wherein, femto-second laser and bias generator, lock-in amplifier and fibre delay line
All integrated to be placed in all -fiber terahertz time-domain spectroscopy system host, terahertz sources head and Terahertz probe are one integrated
The Terahertz module of photoconductive antenna, hypersphere high resistant silicon lens and package casing.
90 ° of off axis paraboloidal mirrors in this programme, it is four pieces and protects the aspherical of golden film by matrix, reflecting surface plating of duralumin
Reflective optical devices, for forming 8f optical systems, wherein paraboloidal mirror 05 is used for Terahertz caused by terahertz sources head
The collimation of pulse, paraboloidal mirror 03 are used to converge to terahertz pulse in ATR prisms, and paraboloidal mirror 04 is used to collect from ATR
The terahertz pulse of prismatic reflection, paraboloidal mirror 06 are used for the hypersphere high resistant that terahertz pulse is focused on to terahertz detection head
In silicon lens.
Reference ATR prisms and sample ATR prisms in this programme, are one piece and are based on High Resistivity Si（Refractive index is about 3.42,
Resistivity>2 kΩ.cm）The total reflection prism of making, for carrying out the survey of reference light in terahertz time-domain decay total reflection spectrum
Amount；Wherein being made in sample ATR prisms upper surface has cell culture chamber, for being tested the culture of attached cell sample.
Reflecting microscope in this programme, pattern and physiology for the attached cell in real-time monitored cell culture fluid
State, its multiplication factor>It is 200 times, operable apart from fine adjustment, and there is overhead illumination function, it is obtained micro-
Image can be stored, shown and handled by the related software installed on computer.
The machinery for being used for optical element positioning, clamping and light path protection that mechanical structure in this programme makes for aluminium alloy
Structure, its inner hollow, have for off axis paraboloidal mirror and terahertz sources head and detecting head positioning and fixed structure, and
Two windows are provided with rail attachment point, are transmitted for terahertz wave beam.The V-groove that piece stainless steel of guide rail makes, its V-groove
Match with ATR prism bottom shapes, surface processing accuracy is better than 0.1 mm, refers to ATR prisms and sample ATR during for measuring
The slip of prism, in addition, guide rail is also used for connecting two parts mechanical structure, its width and off axis paraboloidal mirror focal length and ATR ribs
The correlations such as mirror dimensional parameters.
All -fiber terahertz time-domain spectroscopy system host in this programme, it is one and includes optical fiber femtosecond laser, lock phase
Integrated system including amplifier, fibre delay line, data acquisition and processing (DAP) board, optical fiber and cable etc., it sends out with Terahertz
Penetrate head, detecting head is combined, using the related software installed on computer can realize terahertz time-domain spectroscopy detection institute it is active
Energy.
As shown in Fig. 2 being provided with cell culture chamber on the surface of sample ATR prisms, culturing room is one and is pasted onto sample
" fence " structure of ATR prisms upper surface, the material for making fence are corrosion-resistant and high temperature（>200°C）Macromolecule material
Material, its height is about 10 mm, there is dismountable transparency silica glass cover plate thereon；Adherent layer is located in cell culture room
Portion, the growth of sample ATR prisms upper surface is close to, forms monolayer.It is to be used for cell culture on the top of adherent layer
Nutrient solution.
Put on sample ATR prisms, there is provided a reflecting microscope, passes through the suprasil window on cell culture chamber
Mouthful（Also window can be taken down）And cell culture fluid, the pattern and physiological status of real-time monitored attached cell, its multiplication factor>
It is 200 times, operable apart from fine adjustment, and there is overhead illumination function, its micro-image obtained can pass through computer
The related software of upper installation is stored, shown and handled.
With reference to foregoing description, the system of this programme is at work:
Flown by the optical fiber femtosecond laser output nm wavelength of two-way 1550 in all -fiber terahertz time-domain spectroscopy system host
Second laser pulse, wherein being used for pumping terahertz sources head all the way produces THz wave, another way is used for pumping terahertz detection
First 2 carry out the detection of THz wave.The THz wave of terahertz sources head radiation first passes through off axis paraboloidal mirror 05 and collimated, then by
Off axis paraboloidal mirror 03 is focused in ATR prisms, and the THz wave being emitted from ATR prisms is collected by off axis paraboloidal mirror 04 again
And collimation, then focused on by off axis paraboloidal mirror 06 in terahertz sources head, exported from terahertz sources head and Terahertz
The directly proportional current signal of time domain electric field, into spectroscopic system main frame, carries out the time domain measurement of THz wave by cable transmission.
Conventional cell training method is first according to before experiment, according to finite concentration after cell dissociation is resuspended（Usually
1x106cells/mL）It is seeded on the ATR silicon prisms that sterilization is crossed.37°C、5%CO2Cultivated in cell culture incubator
At night, make cell adherent growth on ATR silicon prisms.
It will be first placed in during measurement with reference to ATR prisms in light path, and measure its terahertz time-domain electric field, then by guide rail by sample
ATR prisms are placed in light path, measure its terahertz time-domain electric field, are carried out using the reference light and sample light Terahertz electric field of acquisition
FFT, its respective complex frequency domain electric field is obtained, obtained with sample optical frequency domain electric field than reference light frequency domain electric field
System complex transfer function, solved in conjunction with plural Fresnel equation, you can obtain the complex dielectric constant of cell sample
（Or complex index）, by analyzing the change of complex dielectric constant and combining microscopic result, you can to cell sample
Physiological change process spread research.
The invention is not limited in foregoing embodiment.The present invention, which expands to, any in this manual to be disclosed
New feature or any new combination, and disclose any new method or process the step of or any new combination.
Claims (8)
- A kind of 1. experimental provision that living cells based on terahertz time-domain decay total reflection spectrum monitors in real time, it is characterised in that bag Include：For optical element positioning and the mechanical platform clamped, guide rail is provided with the mechanical platform, is set on the guide rail There is the sample ATR silicon prism that can be slided along guide rail and refer to ATR prisms, set on the sightingpiston of the sample ATR silicon prisms It is equipped with culturing room,The guide rail both sides are respectively arranged with one group of off axis paraboloidal mirror, and the off axis paraboloidal mirror of side is used for terahertz sources The light of head is pooled to the ATR prism regions on guide rail, and the off axis paraboloidal mirror of opposite side is used to terahertz light being pooled to terahertz Hereby pop one's head in,Reflecting microscope is provided with above Terahertz light path on the guide rail, on real-time monitored sample ATR prisms Living cells changes,The sample ATR silicon prism, with reference to ATR prisms pass through optical fiber or cable connection to system host.
- 2. the reality that a kind of living cells based on terahertz time-domain decay total reflection spectrum according to claim 1 monitors in real time Experiment device, it is characterised in that shown sample ATR silicon prism and with reference to ATR prisms be based on High Resistivity Si make total reflection rib Mirror, the refractive index of the prism is 3.42, and resistivity is more than 2 k Ω .cm.
- 3. the reality that a kind of living cells based on terahertz time-domain decay total reflection spectrum according to claim 1 monitors in real time Experiment device, it is characterised in that the culturing room on the sample ATR silicon prisms is pasted onto sample ATR silicon prism surfaces including one Fence structure, the Fence structure form the seal cavity of a upper end open with sample ATR silicon prism surface.
- 4. the reality that a kind of living cells based on terahertz time-domain decay total reflection spectrum according to claim 3 monitors in real time Experiment device, it is characterised in that the opening of the seal cavity is provided with the quartz window that can be picked and placeed, and the quartz window is Transparent window.
- 5. the reality that a kind of living cells based on terahertz time-domain decay total reflection spectrum according to claim 1 monitors in real time Experiment device, it is characterised in that one group of off axis paraboloidal mirror includes two 90 ° of off axis paraboloidal mirrors, and two groups four 90 ° off-axis Paraboloidal mirror and Terahertz probe, terahertz sources head form 8f integrated forms terahertz time-domain decay total reflection measurement optical system System.
- 6. the reality that a kind of living cells based on terahertz time-domain decay total reflection spectrum according to claim 1 monitors in real time Experiment device, it is characterised in that the guide rail is a V-groove being made by metal material, and the V-groove is used to support sample Product ATR prisms and reference ATR prisms.
- 7. the reality that a kind of living cells based on terahertz time-domain decay total reflection spectrum according to claim 6 monitors in real time Experiment device, it is characterised in that the bottom of the sample ATR prisms and reference ATR prisms is V-type shape, mutual with the V-groove of guide rail Matching.
- 8. a kind of living cells based on terahertz time-domain decay total reflection spectrum according to claim 6 or 7 monitors in real time Experimental provision, it is characterised in that on shown guide rail the left and right sides be respectively arranged with a window, for passing through for light path.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109060729A (en) * | 2018-10-29 | 2018-12-21 | 中国人民解放军陆军军医大学第附属医院 | Transwell detection device and method based on Terahertz Technique of Attenuated Total Reflectance |
| CN109593652A (en) * | 2018-12-24 | 2019-04-09 | 中国人民解放军军事科学院军事医学研究院 | The vertical exposure system of THz wave for cell experiment |
-
2017
- 2017-09-08 CN CN201721148688.3U patent/CN207163909U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109060729A (en) * | 2018-10-29 | 2018-12-21 | 中国人民解放军陆军军医大学第附属医院 | Transwell detection device and method based on Terahertz Technique of Attenuated Total Reflectance |
| CN109060729B (en) * | 2018-10-29 | 2019-08-20 | 中国人民解放军陆军军医大学第一附属医院 | Transwell detection device and method based on Terahertz Technique of Attenuated Total Reflectance |
| CN109593652A (en) * | 2018-12-24 | 2019-04-09 | 中国人民解放军军事科学院军事医学研究院 | The vertical exposure system of THz wave for cell experiment |
| CN109593652B (en) * | 2018-12-24 | 2023-09-26 | 中国人民解放军军事科学院军事医学研究院 | Terahertz wave vertical exposure system for cell experiment |
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