CN104122228A - A microscopic imaging system for analysis of integrated light interference and scattering information and a method - Google Patents
A microscopic imaging system for analysis of integrated light interference and scattering information and a method Download PDFInfo
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Abstract
A microscopic imaging system for analysis of integrated light interference and scattering information is disclosed and is based on a coaxial Michelson interference light path and a Mirau interference device. A sample in at an objective table that is a half mirror. A reflected light beam generates interference on the Michelson interference light path through the Mirau interference device, an amplified interference image is obtained by a microscope, and phase information of the sample can be extracted from the image. At the same time, after the forward transmission light beam passing through the sample passes through the half mirror, forward small-angle and large-angle scattering are generated. The scattering image can be received by a forward CCD, forward scattering amplitude distribution can be obtained from the scattering image, and the size of the sample can be deconstructed by utilization of the scattering theory. Through integrated phase and scattering information, the system and the method can detect the morphological structure of the sample more accurately. The system and the method have important practical value in the field of microscopic morphological measurement of transparent objects, particularly in the fields of biological cell substructure morphological detection and application.
Description
Technical field
The invention belongs to biological cell ortho states micro-imaging technique field, be specifically related to the micro imaging system of a kind of comprehensive reception and analysis interference information and scattered information.
Background technology
Although it is water white that most cells is usually considered to, but strictly speaking, can not be completely transparent, can not be pure-phase object, therefore when after light transmission cell, the amplitude of light and phase place all can change, and see through the information that the amplitude of light of cell and phase place have all comprised cell.Phase imaging detection technique is from cell to going to obtain the information of cell the amount of phase modulation of incident light wave, light scattering testing technology is from cell, the amount of amplitude modulation of incident light to be started with and obtained the information of cell.Therefore in theory, what phase imaging detection technique and light scattering testing technology were studied is two aspects that see through the optical information of cell, not only separate but also connect each other, complements one another.
Existing most of phase imaging detection technique is all based on interferometric method, can realize the whole audience imaging of object, not only its transverse spatial resolution is high, the general restriction that is only subject to diffraction limit, and its axial resolution is high, can reach sub-wavelength magnitude, be applicable to the detection without scatterer or weak scattering object.Whether there is angle according to two interference lights (object light ripple and reference light wave) recording plane, can be divided into coaxial-type and from shaft type.Typical coaxial quantitative phase imaging technology comprises the Fourier phase microscopy (FPM) that Gabriel Popescu professor research group proposes and grows up for 2004, the parallel coaxial holographic phase microscopy being proposed by the Adam Wax of Duke University professor research group, etc., coaxial quantitative phase imaging technology has good stability, can suppress certain phase noise, but generally need to repeatedly measure; Typically comprise Hilbert phase place microscopy (HPM) from axle quantitative imaging technique, diffraction phase microscopy (DPM), two step phase shift binary channels are interfered etc., and this type of imaging technique image taking speed is fast, but needs large broadband, CCD space.In recent years, Adam professor Wax has proposed slightly to interfere (Slightly Off-Axis Interference) from axle, this technology is between interfering between coaxial interference from axle, compare from axle and interfere and need less broadband, CCD space, comparing coaxial interference needs measurement still less, has optimized spatial resolution.Within 2006, Gabriel Popescu professor research group proposes diffraction phase microscopy (DPM), it combines from the advantage of axial light path and coaxial light path: have good stability and high acquisition rate, but due to the reason of light source, there is speckle in the image of diffraction phase microtechnic (DPM), this will reduce the susceptibility of space phase, and limit its application in ultrastructure research, within 2011, be to propose spatial light interference technique (SLIM) by Gabriel Popescu professor research group to adopt white light to overcome speckle effect as light source equally, but this Technology Need is each quantitative phase figure gathers four width intensity images.Afterwards, Gabriel Popescu research groups in 2012 propose to do with white light the diffraction phase microtechnic (wDPM) of light source, with and the patented technology US 2014/0085715A1 (Diffraction phase microscopy with white light: the diffraction phase microtechnic of white light source) that delivers for 2014 realized the sensitivity on single shot and high time and space.But above-mentioned technology is all to obtain the interference information of sample, only can detect transparent phase place body sample, the substructure of sample is remained in detection difficult.
Light scattering testing technology does not generally need to interfere, what directly measure is scatter intensity distribution, the decay of scattared energy distribution or light intensity etc., the information that it provides is the mean value in measurement volumes, equalization the contribution of single composition to scattering, sample substructure form is larger to its diffuse transmission influence, so there is being important application aspect cell size measurement, as patented technology WO 2014/084957 A1 (Optical system, apparatus and method for flow-cytometry: the optical system of flow cytometer, apparatus and method), but, the quantitative measurment that this type of technology changes for cellular morphology, just analyze from scattering aspect, do not relate to interference information.
The deficiency that the present invention exists mainly for above-mentioned two class technology, has invented the device that can simultaneously receive sample light scattering and the micro-information of phase place, applies this device and can calculate according to different model and algorithms, finally can obtain the ultrastructure form result of pinpoint accuracy.
Summary of the invention
The object of the present invention is to provide a kind of Integrated Light is interfered and scattered information is analyzed micro imaging system and method, enable interference information and the scattered information of comprehensive object analysis, more accurately obtain the substructure form of object.
In order to solve above technical matters, the concrete technical scheme that the present invention adopts is as follows:
The micro imaging system that Integrated Light is interfered and scattered information is analyzed, is characterized in that comprising: coaxial Michelson interference light path and meter Lao interference device, be specially:
Described rice labor interference device is made up of the 3rd lens (7), reflective mirror (8), the second spectroscope (9); After microscope (14) amplifies, be that CCD-2 (16) be received as digit phase image and by computing machine (17) carry out numerical evaluation and processing by interference image receiver through the image of described rice labor interference device;
Described Michelson interference optical routing light source (1), the first spectroscope (6), reflective mirror (8), the second spectroscope (9), objective table (10) composition; Also comprise the beams extended by lens colimated light system, the scattering strength receiver that are formed by the 4th lens (11) and the 5th lens (12) be CCD-1 (13), interference image receiver be CCD-2 (16), microscope (14), the 6th lens (15), for scattered information and phase information being gathered to the computing machine (17) of Storage and Processing;
Described objective table (10) is a semi-transparent semi-reflecting lens that angle is adjustable, adjustable with reflective mirror (8) angle, can realize coaxial, from axle and the slight optical path adjusting from axle interference;
Described objective table (10) is semi-transparent semi-reflecting lens, to ensure that can see through objective table through the scattered light of sample transmits downwards;
The first spectroscope (6) and surface level are in angle of 45 degrees; The putting position of the second spectroscope (9) ensure to make the reflected light of objective table (10) and the reflected light of reflective mirror (8) the distance of process equate; Reflective mirror (8) is placed on the 3rd lens (7) center below, and its size is the smaller the better on the basis that can ensure to occur reflection.
According to the micro imaging method of the micro imaging system of described a kind of Integrated Light interference and scattered information analysis, it is characterized in that comprising the following steps:
Step 1, after the beam shaping system of the light beam that makes light source (1) via first lens (2), field stop (3), the second lens (4), aperture diaphragm (5) composition, transfer to the first spectroscope (6); The reflected light of described the first spectroscope (6) is transferred to the second spectroscope (9) by the 3rd lens (7), light beam is divided into two parts by the second spectroscope (9): it is upper that a part reflexes to reflective mirror (8), and a part is transmitted on sample; Be transmitted to light beam on sample when objective table (10), a part reflects to obtain reflected light, described reflected light again by after sample and the reflected light of reflective mirror (8) meet and produce interference, after elder generation, through the 3rd lens (7), the first spectroscope (6), microscope (14) and the 6th lens (15), above form micro-interference image at CCD-2 (16); Described micro-interference image as calculated machine (17) obtains reflecting the PHASE DISTRIBUTION information of sample morphosis after processing;
Step 2, utilize the second spectroscope (9) be transmitted to light on sample when objective table (10) part light transmission objective table (10) transmitted light; Described transmitted light comprises the scattered information of sample, through the beams extended by lens colimated light system of the 4th lens (11), the 5th lens (12) composition, described scattered information is received as digital picture and is carried out numerical evaluation by computing machine (17) by CCD-1 (13) and obtains scattering strength and process to obtain the scattering amplitude distributed intelligence of reflection sample size;
Step 3, the scattered information that the interference information that computing machine (17) obtains described step 1 and step 2 obtain, carries out calculating based on scattering theory and phase imaging theory, finally can obtain phase place body sample form, structure and size information.
principle of work of the present invention is as follows:
The light beam being sent by light source 1, transmit forward via first lens 2, field stop 3, the second lens 4, after the beam shaping system that aperture diaphragm 5 forms, transfer to the first spectroscope 6, the reflected light of described the first spectroscope 6 continues to propagate downwards, transfer to the second spectroscope 9 through the 3rd lens 7, light beam is divided into two parts by described the second spectroscope 9, a part reflexes on reflective mirror 8, a part is transmitted on sample: the light part being transmitted on sample reflects, the reflected light of this part reflected light and reflective mirror 8 meets and interferes at the second spectroscope 9 places, afterwards through the 3rd lens 7, the first spectroscope 6, microscope 14, the 6th lens 15, receive micro-interference pattern by CCD-2 16, be transmitted to some objective table through semi-transparent semi-reflecting lens of light on sample, the beams extended by lens colimated light system forming through the 4th lens 11, the 5th lens 12, collects sample scattering strength information by CCD-1 13.Last computing machine 17, to the interference information obtaining and scattered information, is carried out calculating based on scattering theory and phase imaging theory, finally can obtain phase place body sample form, structure and the size information of pinpoint accuracy.
the present invention has beneficial effect.Light source of the present invention adopts low relevant white light source, improves resolution, reduces noise; The present invention adopts coaxial Michelson interference light path, has improved the stability of experiment; Objective table of the present invention is the adjustable semi-transparent semi-reflecting lens of an angle, and the scattered light that can ensure to comprise sample scattering information can see through objective table and continue transmission, and adjustable with reflective mirror angle, can realize coaxial, from axle and the slight optical path adjusting from axle interference.This is utilized CCD-1 13 collected specimens scattering strengths, CCD-2 16 to receive micro-interference image, makes computing machine can comprehensively analyze scattered information and interference information, obtains having phase place body sample form, structure and the size information of pinpoint accuracy; After microcobjective of the present invention is positioned at interference system, can reduce phase noise.Therefore, system applies of the present invention is wide, has very strong using value.
Brief description of the drawings
Fig. 1 is light path schematic diagram corresponding to micro imaging system that Integrated Light of the present invention is interfered and scattered information is analyzed.
In figure: 1. light source; 2. first lens; 3. field stop; 4. the second lens; 5. aperture diaphragm; 6. the first spectroscope; 7. the 3rd lens; 8. reflective mirror; 9. the second spectroscope; 10. objective table; 11. the 4th lens; 12. the 5th lens; 13.CCD-1; 14. microscopes; 15. the 6th lens; 16.CCD-2; 17. computing machines.
Embodiment
Below in conjunction with accompanying drawing, technical scheme of the present invention is described in further details.
The micro imaging system that Integrated Light of the present invention is interfered and scattered information is analyzed, realizes by coaxial Michelson's light path and meter Lao interferometer, as shown in Figure 1.
The light beam being sent by light source 1, transmit forward through first lens 2, field stop 3, the second lens 4, the beam shaping system that aperture diaphragm 5 forms, arrive the first spectroscope 6, the reflected light of described the first spectroscope 6 transfers to the second reflective mirror 9 through the 3rd lens 7, light beam is divided into two parts by the second spectroscope 9, a part reflexes on reflective mirror 8, a part is transmitted on sample: the light part being transmitted on sample reflects, the reflected light of this part reflected light and reflective mirror 8 meets and interferes at the second spectroscope 9 places, afterwards through the 3rd lens 7, the first spectroscope 6, microscope 14, the 6th lens 15, receive micro-interference image by CCD-2 16, and be stored in computing machine 17, the light being transmitted on sample also has part to see through the objective table 10 continuation transmission downwards of semi-transparent semi-reflecting lens, and the beams extended by lens colimated light system forming through the 4th lens 11, the 5th lens 12 gathers scattering strength, and is stored in computing machine 17 on CCD-1 13.Last computing machine 17, according to the sample micro-interference image and the scattering strength information that obtain, is carried out calculating based on scattering theory and phase imaging theory, finally can obtain phase place body sample form, structure and size information.
Claims (2)
1. the micro imaging system that Integrated Light is interfered and scattered information is analyzed, is characterized in that comprising: coaxial Michelson interference light path and meter Lao interference device, be specially:
Described rice labor interference device is made up of the 3rd lens (7), reflective mirror (8), the second spectroscope (9); After microscope (14) amplifies, be that CCD-2 (16) be received as digit phase image and by computing machine (17) carry out numerical evaluation and processing by interference image receiver through the image of described rice labor interference device;
Described Michelson interference optical routing light source (1), the first spectroscope (6), reflective mirror (8), the second spectroscope (9), objective table (10) composition; Also comprise the beams extended by lens colimated light system, the scattering strength receiver that are formed by the 4th lens (11) and the 5th lens (12) be CCD-1 (13), interference image receiver be CCD-2 (16), microscope (14), the 6th lens (15), for scattered information and phase information being gathered to the computing machine (17) of Storage and Processing;
Described objective table (10) is a semi-transparent semi-reflecting lens that angle is adjustable, adjustable with reflective mirror (8) angle, can realize coaxial, from axle and the slight optical path adjusting from axle interference;
Described objective table (10) is semi-transparent semi-reflecting lens, to ensure that can see through objective table through the scattered light of sample transmits downwards;
The first spectroscope (6) and surface level are in angle of 45 degrees; The putting position of the second spectroscope (9) ensure to make the reflected light of objective table (10) and the reflected light of reflective mirror (8) the distance of process equate; Reflective mirror (8) is placed on the 3rd lens (7) center below, and its size is the smaller the better on the basis that can ensure to occur reflection.
2. according to the micro imaging method of the micro imaging system of a kind of Integrated Light interference described in claim and scattered information analysis, it is characterized in that comprising the following steps:
Step 1, after the beam shaping system of the light beam that makes light source (1) via first lens (2), field stop (3), the second lens (4), aperture diaphragm (5) composition, transfer to the first spectroscope (6); The reflected light of described the first spectroscope (6) is transferred to the second spectroscope (9) by the 3rd lens (7), light beam is divided into two parts by the second spectroscope (9): it is upper that a part reflexes to reflective mirror (8), and a part is transmitted on sample; Be transmitted to light beam on sample when objective table (10), a part reflects to obtain reflected light, described reflected light again by after sample and the reflected light of reflective mirror (8) meet and produce interference, after elder generation, through the 3rd lens (7), the first spectroscope (6), microscope (14) and the 6th lens (15), above form micro-interference image at CCD-2 (16); Described micro-interference image as calculated machine (17) obtains reflecting the PHASE DISTRIBUTION information of sample morphosis after processing;
Step 2, utilize the second spectroscope (9) be transmitted to light on sample when objective table (10) part light transmission objective table (10) transmitted light; Described transmitted light comprises the scattered information of sample, through the beams extended by lens colimated light system of the 4th lens (11), the 5th lens (12) composition, described scattered information is received as digital picture and is carried out numerical evaluation by computing machine (17) by CCD-1 (13) and obtains scattering strength and process to obtain the scattering amplitude distributed intelligence of reflection sample size;
Step 3, the scattered information that the interference information that computing machine (17) obtains described step 1 and step 2 obtain, carries out calculating based on scattering theory and phase imaging theory, finally can obtain phase place body sample form, structure and size information.
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