CN1826518A - System and method for measuring phase - Google Patents

Abstract

Preferred embodiments of the present invention are directed to systems for phase measurement which address the problem of phase noise using combinations of a number of strategies including, but not limited to, common-path interferometry, phase referencing, active stabilization and differential measurement. Embodiment are directed to optical devices for imaging small biological objects with light. These embodiments can be applied to the fields of, for example, cellular physiology and neuroscience. These preferred embodiments are based on principles of phase measurements and imaging technologies. The scientific motivation for using phase measurements and imaging technologies is derived from, for example, cellular biology at the sub-micron level which can include, without limitation, imaging origins of dysplasia, cellular communication, neuronal transmission and implementation of the genetic code. The structure and dynamics of sub-cellular constituents cannot be currently studied in their native state using the existing methods and technologies including, for example, x-ray and neutron scattering. In contrast, light based techniques with nanometer resolution enable the cellular machinery to be studied in its native state. Thus, preferred embodiments of the present invention include systems based on principles of interferometry and/or phase measurements and are used to study cellular physiology. These systems include principles of low coherence interferometry (LCI) using optical interferometers to measure phase, or light scattering spectroscopy (LSS) wherein interference within the cellular components themselves is used, or in the alternative the principles of LCI and LSS can be combined to result in systems of the present invention.

Description

The system and method that is used for phase measurement

Technical field

This part application is a U.S. Patent application the 10/823rd, the follow-up application of part of No. 389 (submissions on April 13rd, 2004), and the 10/823rd, No. 389 patent is the 10/024th, the follow-up application of No. 455 part (submission on Dec 18 calendar year 2001), and require the United States Patent (USP) provisional application the 60/479th, No. 732 rights and interests of (submission on June 19th, 2003).The full content of above-mentioned application is intactly incorporated into by quoting as proof at this.

Background technology

Needing to be widely used in the optical distance measurement of sub-wavelength range-sensitivity based on the optical interferometry technology of phase place.Optical range is defined as the product of refractive index and length.Yet most of such technology are limited in 2 π blur level or integer ambiguities of the difficulty of interferogram aspect away from each other that can be defined as narrating axial scan by well-known problems in this field.Low coherence's interferometry (LCI) based on unmodified harmonic phase can be used for determining difference optical range (n _{λ 2}-n _{λ 1}) L, wherein L is an actual range, n _{λ 1}And n _{λ 2}It is the wavelength X separately ₁, λ ₂Under refractive index, superpose tracked if optical range increases the poor phase potential energy measured with the LCI that applies gradually by its 2 π.In order to determine (the n of DNA in the solution _{λ 2}-n _{λ 1}), for instance, DNA concentration increases in measuring test tube gradually.The metering system of even now is operate as normal in controlled environment, but it can't be realized under the lower situation of the operability of sample.For example, this method is inoperative to the fixing slab material that is forced to complete preservation.

Problem is interferogram that unmodified LCI can not narrate axial scan this fact away from each other, is described to 2 π blur level problems at this.This is the problems of puzzlement great majority based on the optical interferometry technology of phase place.Therefore, these technology can not fully be determined optical range.So most of such technology are used to such as the structure of calculating continuous surface or survey application the variable in distance that changes in time, wherein phase unwrapping by between the consecutive point or the phase place on little time increment relatively be possible.

In many application, importantly measure to see through sample quantitatively or from the phase place of the light wave of sample reflection.Specifically, see through biological sample or from the phase place light wave of the light wave of biological sample reflection can live or abiotic cell form effective 26S Proteasome Structure and Function probe.

Interferometry is the broad-spectrum technology that is used for measuring the phase place of light wave.A common issue with of quantitative interferometry is the susceptibility to the phase noise that causes owing to the outside perturbation such as vibration, air movement and thermal drift.Therefore, still need to solve the phase measuring system of phase noise problem.

Interferometry is a kind of approach that obtains the phase information that is associated with sample.Technology such as phase correlation and Nomarski microscope is used optical phase as just the contrast key element, and the quantitative information about its quantity is not provided.Exist some to be used for measuring the almost technology of the phase place of the light wave of transparent sample that sees through.These comprise digital recording type interference microscope (DRIMAPS) and the non-interferometer of the PHASE DISTRIBUTION figure that transmits via the intensity equation is surveyed.

The reflection interference mensuration can have the little a lot of sensitivity of wavelength than used light wave.Measurement by part nanometer or smaller scale is being common aspect metrology and the microtexture sign.Yet, such as biological cell and tissue on the faint reflection sample with the work of having finished of nano level interferometry seldom.Optical coherence tomography (OCT)---use biological sample interfere measurement technique---mainly is relevant with amplitude rather than with relevant from the phase place of reflected light wave interference, so aspect resolution, be limited to the coherent length of used light wave, normally the 2-20 micron.

The reflection interference mensuration of quoting phase place has been used for measuring the volume change of cell monolayer.The used interferometer based on harmonic phase needs two light sources, is low relatively (5 hertz), and the phse sensitivity of about 20mrad is arranged in described bandwidth.Therefore, the effective phase measuring system that still needs to solve the phase noise problem and help the different imaging applications of exploitation.

Summary of the invention

The preferred embodiments of the invention relate to the phase measuring system of the problem of processing such as phase noise, for example, use to include but not limited to altogether to light path interferometry, phase place reference, initiatively some kinds of tactful combinations of stable and variate.Embodiment relates to the optical devices that make tissue or little biosome imaging with light wave.These embodiments can be applied to, for example, and stechiology and Neuscience field.Described embodiment preferred is based on the principle of phase measurement and imaging technique.Use the science motivation of phase measurement and imaging technique to originate from, for example, the cell biology of pattern of sub-micron level, it can comprise dysplasia, cell communication, neuron transmission without restriction and use the imaging source of the program implementation of genetic code.The structure of subcellular fraction component and dynamics can not use existing method and technology (comprise, for example, X ray and neutron scattering) to study in their state of nature now.Otherwise the technology based on light wave of nanometer resolution can be studied the cell machine in its state of nature.Therefore, the preferred embodiments of the invention comprise the system based on the principle of interferometry and/or phase measurement, and are used to study stechiology.These systems comprise low coherence's interferometry (LCI) of using the optical interdferometer Measurement Phase, or wherein use the principle of the interference light scattering of wave spectroscopy (LSS) within the cell component itself, or in replacement scheme, the principle of LCI and LSS can be combined and cause system of the present invention.

The preferred embodiment of phase measurement and imaging system comprises initiatively stable interferometer, isolates intervention instrument, altogether to the light path interferometer, and can comprise the phasecontrast microscope that the usage space light wave is modulated.

In preferred embodiments, method of the present invention relates to preferably with inferior nano-precision, based on the accurate phase measurement technology of long optical range arbitrarily.The preferred embodiments of the invention are used the interferometer of light source that harmonic relationships is arranged (continuous wave (CW) and the secondary light source of low coherence (LC) is arranged), for example, and the Michelson interferometer.Low coherence source provides broad spectral bandwidth, and preferably, bandwidth is greater than 5nm with regard to the wavelength of 1 micron (μ m), and for example, essential bandwidth can be along with wavelength and application change.Centre wavelength by fine setting low-coherence light source between the scanning of target sample can be used for the separation between the inferior nanometer accuracy measurement reflecting interface in the phase relation between the heterodyne signal of CW and low coherence's light wave.Because this method is not perplex problem---the 2 π blur leveles of great majority based on the technology of phase place fully, so can be used under the situation that does not reduce precision, measuring long arbitrarily optical range.The application of the preferred embodiment of method of the present invention is a refractive index of accurately determining sample under the setted wavelength of the sample of known actual (real) thickness.The Another application of the preferred embodiment of method of the present invention is accurately to determine the actual (real) thickness of sample with known refractive index.The further application of the preferred embodiment of method of the present invention is accurately to determine the refractive index ratio under two given wavelength.

In other possible preferred embodiment, low-coherence light source provides bandwidth enough wide light wave, is preferably greater than 5nm, so that the first low relevant wavelength and second that provides centre wavelength separately to be separated from each other more than about 2nm simultaneously hangs down relevant wavelength.The frequency spectrum of described low relevant wavelength is overlapping deficiently.Additional detector and wave filter are arranged in the interferometer so that transmission and survey two low relevant wavelength.

The method of preferred embodiment can be used for carrying out accurate optical distance measurement.According to such measurement result, the optical mass-energy of target object is accurately measured.By the dispersion profile figure of measurement target, structure and/or chemical property that can estimating target.This dispersion profile figure plots figure to the index difference under the various wavelength.In biomedical background, the preferred embodiments of the invention are accurately determined the chromatic dispersion character of biological tissue by noncontact and non-invasive mode.Described dispersion measurement can be used on eye's cornea or the aqueous humor.The sensitivity that is realized is enough to survey the optical change fixed according to concentration of glucose.In the preferred embodiment of the inventive method, the glucose level of blood can be determined by the aqueous humor of non-intrusive measurement eyes and/or the dispersion profile figure of vitreum or cornea.The preferred embodiments of the invention can be applied to measuring the little feature that forms during integrated circuit and/or the optoelectronic components making as the measuring technique in the semiconductor manufacturing.Because the preferred embodiment of described method be noncontact with nondestructive, so can when making semiconductor structure or optics, monitor their thickness.

Use the preferred embodiment of Mach-Zender heterodyne system interferometer according to the present invention, be used for measuring first wavelength that comprises the steps: to provide light wave through the method for the phase place of the light wave of a part of sample; Guide the light wave of first wavelength along first light path and second light path, first light path extends to the change of on the sample medium that will measure and second path experience path aspect, and survey from the vectorial light wave of sample and from the light wave of second light path so as to measure light wave by two are separated on the sample medium point in the change aspect the phase place.Medium comprises biological tissue, for example, and neuron.This method comprises to be used photodiode array or makes the phase place of sample in the imaging simultaneously of numerous positions with the fibre bundle of photodiode coupling.This method further is included in the described light wave of frequency displacement in second light path.This method comprises He-Ne Lasers source () or the low-coherence light source that emission first wavelength is provided.

According to another aspect of the present invention, initiatively stable interferometer is used to measure the method for the phase place of the light wave by a part of sample, this method comprises the steps: to provide first signal and the secondary signal that is produced by first light source and secondary light source respectively, and secondary light source is low coherence source.This method comprises along first light path and second light path and guides first signal and secondary signal; Change the path length difference between first light path and second light path; Produce the output signal that indication has the first and second signal sums of optical path delay therebetween; In this output signal of interferometer locking modulating frequency modulated; And the phase place of making progress to determine sample by the time of interferometer locking phase.First and second signals are two low coherence's signals.This method further comprises with frequency mixer or lock-in amplifier demodulation first signal.This method comprises with electronics method generation interferometer locking phase.

According to another aspect of the present invention, the twin-beam reflection interferometer is used to measure the system through the phase place of the light wave of a part of sample.This system comprises first light source that produces first signal; Produce by time delay and separate the interferometer of the secondary signal of two pulses with first signal; From first light path of interferometer and sample relation and second light path of getting in touch from interferometer and reference field; And according to respectively from first and second signals of sample and reference field with from the detector system of interferometry first heterodyne signal between the light wave of sample and reference field reflection.This system comprises the phase place of detection indication sample reflection with respect to the heterodyne signal of the phase place of reference field reflection.First signal is low coherence's signal.First source of light wave can comprise one of superluminescent diode or multimode laser diode without restriction.Second path of interferometer further comprises first path and second path, and there is acousto-optic modulator in second path.This system comprises the light path that comprises optical fiber.This system comprises the heterodyne Michelson interferometer of shock insulation.This interferometer further comprises the catoptron that is attached to translation stage adjusted path length difference.Described detection system comprises first detector and detection second detector from the signal of reference field reflection of detection from the signal of sample reflection.

According on the other hand, the invention provides and use phasecontrast microscope and the modulation of space light wave to make the sample imaging method.In various embodiment, described method comprises and illuminates sample owing to come from the light wave that illuminates sample low frequency space component and high frequency spatial component are arranged.The phase place of low frequency space component is offset so that provide three by the low frequency space component of phase shift at least.Preferably, phase place be by, for example, the increment of pi/2 skew so that produce the low frequency space component of phase shift pi/2, π and 3 pi/2s.

Unmigrated low frequency space component and at least three along separately interfering the high frequency spatial component to light path altogether, are produced strength signal that each is separately interfered by the low frequency space component of phase shift.Then, for example, use at least four strength signals to produce the image or the phase image of sample.

According on the other hand, the invention provides the method that the non-contact optical measurement has the sample of reflecting surface, this method has the following steps: first light source that produces first signal is provided; Use double beam interferometer to produce the secondary signal of separating two pulses by the time delay and first signal; Provide from first light path of interferometer and sample relation and second light path of getting in touch from interferometer and reference field; Foundation is respectively from first and second signals of sample and reference field with from interferometry first heterodyne signal between the light wave of sample and reference field reflection; And survey and indicate the phase place of sample reflection with respect to the heterodyne signal of the phase place of reference field reflection.

In preferred embodiments, first signal is low coherence's signal.First light source may be superluminescent diode or multimode laser diode.Described interferometer further comprises first path and second path, and there is acousto-optic modulator in second path.Described method further comprises the light path that comprises optical fibers.Described sample may be the part of neurocyte.

In preferred embodiments, described interferometer comprises the heterodyne system Michelson interferometer of shock insulation.This interferometer further comprises and is attached to the catoptron of controllably regulating path length difference on the translation stage.Embodiment preferred comprises that the heterodyne system of the measurement of finishing the non-contacting and first kind of interferometry of neural first kind of expanding hangs down coherence's interferometer.The neural biophysics mechanism that expands can be used other aixs cylinder imaging and analysis according to the preferred embodiments of the invention.The low coherence's interferometer of twin-beam is having many other application aspect the nanoscale motion of measuring living cells.Other embodiment can comprise with the interferometer being the microscope that the machinery variation that single neuron is associated with action potential is surveyed on the basis.Relevant interferometric method also is used for measuring and is being changed by cell volume in the cell monolayer of cultivating.

Another aspect of the present invention comprises the fibre-optical probe that is used for making the sample optical imagery, and this fibre-optical probe includes the shell of near-end and far-end; At the near-end of shell and the optical fiber collimator of light source coupling; And at the gradually changed refractive index lens of shell far-end, these lens have first and second surfaces, and wherein first surface is a reference surface, and the numerical aperture of this probe provides from the effective light wave of the scattering surface of sample and assembles.This probe comprises that further the fibre-optical probe that is contained on the parallel-moving type objective table is so that finish one of two-dimensional phase imaging and three-dimensional confocal phase imaging at least.The parallel-moving type objective table comprises the scanning piezoelectric transducer.The numerical aperture of probe is in about scope of 0.4 to 0.5.

The above-mentioned feature with other of the described system and method that is used for phase measurement and advantage will become obvious from the description more specifically of the preferred embodiment of the system and method for the following accompanying drawing illustrated of representing identical part at similar benchmark character at different views everywhere.These pictures needn't be drawn to scale, but emphasize to illustrate principle of the present invention.

Description of drawings

Fig. 1 is the synoptic diagram of preferred embodiment of measuring the system of optical range according to the present invention;

Fig. 2 illustrates the low coherence's heterodyne signal that is associated with reflecting interface according to preferred embodiment, wherein regulates low coherence's wavelength compression or Expansion Interface heterodyne signal (the adjusting direction that depends on the centre wavelength of low-coherence light source) on every side;

Fig. 3 according to preferred embodiment illustrate with sample in two heterodyne signals that reflecting interface is associated, wherein reduce the heterodyne signal around low coherence's wavelength compression interface;

Fig. 4 illustrates the scanning of the sample at two interfaces according to the preferred embodiments of the invention, (a) low coherence's heterodyne signal, and (b) vestige, wherein enlarged drawing is represented the phase place striped, each striped and λ _CWOptical range corresponding, (c) at the vestige at two difference places of Δ, wherein arrow is pointed out phase crossover, Z-axis is unit with the radian;

Fig. 5 illustrates according to the preferred embodiments of the invention and determines to make based on S by selection _PhaseAnd S _FrigEEstimation between (the n of the numerical value actual measurement that minimizes of error _775nmL) correct estimation approach;

Fig. 6 A and 6B illustrate the process flow diagram of measuring the method for optical range according to the preferred embodiments of the invention;

Fig. 7 is the synoptic diagram of other possible preferred embodiment of measuring the system of optical range according to the present invention;

Fig. 8 A and 8B illustrate the process flow diagram of measuring the alternative method of optical range according to the preferred embodiments of the invention;

Fig. 9 schematically illustrates the preferred embodiment based on the system of optical fiber of the thickness of optically transparent material measurement such as glass plate, tissue sample or the organized layer;

Figure 10 illustrates the preferred embodiment that is used for the system of the present invention of vitreum and/or water sample body fluid glucose measurement system according to the present invention;

Figure 11 illustrates initiatively stable Michelson interferometer, is catoptron according to the preferred embodiments of the invention M wherein, and MM is a mobile mirror, BS is a spectroscope, and PM is a phase-modulator, and D is a detector, LO is the local oscillations source, and MX is a frequency mixer, and S is a summing amplifier;

Figure 12 illustrates the stable interferometer that is used for the low coherence's interferometry (LCI) of optical delay phase sensitivity, and wherein DBS is dichroic beam splitters according to the preferred embodiments of the invention;

Figure 13 illustrates the sample demodulated interferential figure at a pair of interface when path length difference Δ L changes according to the preferred embodiments of the invention;

Figure 14 A illustrates the system that is used for the low coherence's interferometry (LCI) of stable phase sensitivity, and wherein LC1 and LC2 are low coherence's light beams according to the preferred embodiments of the invention;

Figure 14 B illustrates according to the present invention and uses piezoelectric transducer to produce the alternate embodiment of the system that is used for the low coherence's interferometry (LCI) of initiatively stable phase sensitivity of phase change;

Figure 15 A and 15B are the bar graphs of the demodulation of LC1 and LC2, and wherein cover glass reflection (big) is represented according to the preferred embodiments of the invention and from the reflection (little) of sample in two of the LC2 signal peaks;

Figure 16 illustrates the imaging system that is used for the low coherence's interferometry of stable phase sensitivity according to the preferred embodiments of the invention;

Figure 17 illustrates the optical design of the expansion that is used for the two-dimensional phase imaging, and wherein according to the preferred embodiments of the invention, solid line is represented incident ray, and dotted line is represented backscattered light;

Figure 18 A illustrates 2 Mach-Zender heterodyne ineterferometers according to the preferred embodiments of the invention;

Figure 18 B illustrates imaging Mach-Zender heterodyne ineterferometer according to the preferred embodiments of the invention;

Figure 18 C illustrates heterodyne signal and the gating signal that is associated with the embodiment of describing with reference to Figure 18 B;

Figure 18 D illustrates imaging twin-beam heterodyne system interferometer according to the preferred embodiments of the invention;

Figure 19 illustrates the low coherence's interferometer of twin-beam heterodyne system of isolation according to the preferred embodiments of the invention;

Figure 20 illustrates the low coherence's interferometer of two reference field heterodyne systems according to the preferred embodiments of the invention;

Figure 21 illustrates the preferred embodiment of optics reference interferometer according to the preferred embodiments of the invention;

Figure 22 schematically illustrates according to the preferred embodiments of the invention owing to be positioned at the component of the actual measurement phase place that causes on the same surface (glass) as the benchmark millet cake of target sample;

Figure 23 A and 23B just illustrate to graphic formula the voltage and the corresponding phase change of piezoelectric transducer (PZT) respectively with reference to the illustrational embodiment of Figure 21 according to the preferred embodiments of the invention;

Figure 24 according to the illustrational interferometer of Figure 21 with the radian be unit graphic formula illustrate noiseproof feature;

Figure 25 A and 25B are the signal expression that is used for the demarcation assembly of sample signal and reference field signal according to the preferred embodiments of the invention;

Figure 26 schematically illustrates the preferred embodiment according to the interferometer system of the preferred embodiments of the invention;

Figure 27 illustrates the synoptic diagram of measuring the system of neural displacement according to the preferred embodiments of the invention;

Figure 28 A and 28B according to the preferred embodiments of the invention graphic formula illustrate neural displacement (nm) and electromotive force (μ V) relevant for the time (ms);

Figure 29 according to the preferred embodiments of the invention graphic formula illustrate spike potential (cross) and displacement (circle), wherein the variable stimulating current amplitude of single nerve;

Figure 30 illustrates the optical design that is used for the scanning system of double beam interferometer according to the preferred embodiments of the invention;

Figure 31 illustrates according to the preferred embodiments of the invention galvanometer position and uses the phase data of Lissajous scanning from the cover glass collection of sky;

Figure 32 A and 32B illustrate respectively according to the preferred embodiments of the invention that use shows and the color map and the retroeflection intensity image of the phase image of the illustrational data of graphic formula in Figure 31;

Figure 33 schematically illustrates the focus issues that solves by the preferred embodiments of the invention;

Figure 34 illustrates the design that is used for bifocal lens according to the preferred embodiments of the invention;

Figure 35 illustrates the alternate design that is used for bifocal lens according to the preferred embodiments of the invention;

Figure 36 illustrates according to the optimum distance between the preferred embodiments of the invention calculating lens f3 (bifocal lens) and the f2;

Figure 37 illustrates according to the preferred embodiments of the invention and makes bifocal lens;

Figure 38 illustrates according to the preferred embodiments of the invention, when object lens retroeflection intensity by the optical circulator measurement when the glass cover slide scans;

Figure 39 illustrates according to the preferred embodiments of the invention, and retroeflection intensity is with the object focal point change in location of using bifocal lens f3;

Figure 40 illustrates according to the preferred embodiments of the invention under the situation of two kinds of cover glass reflections, and retroeflection intensity is with the object focal point change in location of using bifocal lens;

Figure 41 illustrates according to the preferred embodiments of the invention, when the distance between f2 and the f3 is adjusted to and space between the glass surface of front and back when matching, retroeflection intensity is with the object focal point change in location of using bifocal lens under the situation of two kinds of cover glasses reflections;

Figure 42 A and the 42B that are referred to as Figure 42 illustrate owing to axial the coupling in optical system with the light beam edge forms extra less peak according to the preferred embodiments of the invention;

Figure 43 A illustrate according to the preferred embodiments of the invention as a whole element the twin-beam probe on reference field surface is arranged;

Figure 43 B is another preferred embodiment of two-beam interference instrument probe;

Figure 43 C is the image of two nerve fibres;

Figure 43 D is the image of heterodyne signal amplitude as the function of position;

Figure 43 E is the reflected phase will image of the same sample seen in Figure 43 D;

Figure 44 is according to the preferred embodiments of the invention, be applicable to research during the action potential in nerve the sample chart of the geometric twin-beam of observed displacement effect probe;

Figure 45 is used for twin-beam probe system by scanning head or sample imaging according to the preferred embodiments of the invention;

Figure 46 A-46C illustrates according to the preferred embodiments of the invention, uses intensity image, phase image and the bright field image of bifocal twin-beam microscope from the backscattered light wave of human cheek epithelial cell of drying;

Figure 46 D-46G illustrates at the microscopical contour curve of the twin-beam of Figure 43 illustrated and measures ability, wherein Figure 46 D is the intensity image of the core of the illustrational plano-convex lens of Figure 46 E system, Figure 46 F is the phase mapping figure of reflecting light, and Figure 46 G is the xsect of phase image, according to the phase place of the preferred embodiments of the invention by the quadratic fit expansion.

Figure 47 A-47E integrates according to synoptic diagram, phase place stepping and bucket formula that the preferred embodiments of the invention illustrate phase-shifting interference measuring method system respectively;

Figure 48 A-48C illustrates the principle of integrating according to stroboscopic formula difference interference measuring system of the preferred embodiments of the invention and bucket formula respectively;

Figure 49 A illustrates the twin-beam stroboscopic formula heterodyne ineterferometer according to the preferred embodiments of the invention;

Figure 49 B and 49C illustrate performance focuses on the phase noise on the static glass surface according to the twin-beam probe of the preferred embodiments of the invention data;

Figure 50 A illustrates another preferred embodiment, wherein is drawn towards common path from the light wave in the path that separates and is focused on the zones of different of measured material;

Figure 50 B is to use the preferred embodiment of the double-beam system of system shown in Figure 50 A;

Figure 50 C provides the details about the polarization component within the illustrational system of Figure 50 B;

Figure 51 A-51D is that the signal of the various features of this iamge description according to the preferred embodiments of the invention is expressed;

Figure 52 schematically illustrates the various embodiment based on the microscopic system of transmission geometry according to the present invention;

Figure 53 schematically illustrates the various embodiment based on the microscopic system of reflection geometry according to the present invention;

Figure 54 A and the 54B that are referred to as Figure 54 schematically illustrate an embodiment integrating various embodiments of the present invention with optical microscope;

Figure 55 schematically illustrates the various embodiment that the present invention utilizes the system and method for 4-f system;

Figure 56 schematically illustrates an embodiment utilizing the phasecontrast microscope system of space light wave modulation (SLM) according to the present invention;

Figure 57 A and 57B schematically illustrate the photoelectric effect on the pixel of image in amplitude pattern and phase pattern according to the preferred embodiments of the invention;

Figure 58 A-58C is the block scheme according to the various embodiment of the SLM pattern of the preferred embodiments of the invention operation;

Figure 59 is according to the example of the preferred embodiments of the invention at the calibration curve of the instrument acquisition of operating by the amplitude pattern;

Figure 60 A-60D shows the image that uses the system of reflection geometry to obtain according to the preferred embodiments of the invention under four kinds of different phase shifts;

Figure 61 schematically illustrates according to the high frequency waves vector component E of the preferred embodiments of the invention at electromagnetic field vector E and electromagnetic field _HLow frequency wave vector component E with electromagnetic field _LBetween relation;

Figure 62 is the Δ φ image that for example uses the calibration sample that the data in Figure 35 A-35D and equation 55 illustrated produce according to the preferred embodiments of the invention;

Figure 63 is to use the phase image according to the calibration sample of the embodiment 1 of system and method for the present invention;

Figure 64 shows the phase image that uses transmission geometry to obtain according to the preferred embodiments of the invention;

Figure 65 shows the onion cell intensity image that obtains according to the preferred embodiments of the invention;

Figure 66 shows the onion cell phase image that uses transmission geometry to obtain according to the present invention;

Figure 67 illustrates experimental assembly according to embodiment preferred, and wherein VPS is virtual pointolite; CL is a correcting lens; IP is an imaging plane; P is a polarisation ripple mirror; BS is a beam splitter; FL is a fourier transform lens; PPM is programmable phase-modulator; CCD is a charge-coupled image sensor, and PC is a personal computer;

Figure 68 A and 68B illustrate according to what embodiment preferred used that 10 * micro objective obtains and are immersed in experimental result in 100% glycerine about polystyrene microsphere, and wherein Figure 68 A is an intensity image, and Figure 68 B is quantitative phase image.The phase place that the color-bar representative is expressed with nm;

Figure 69 A-69C illustrates the LCPM image that uses 40 * micro objective to obtain according to preferred embodiment, wherein Figure 69 A is the phase image of the mitotic HE1A cancer cell of experience, Figure 69 B is the phase image of whole blood film, and Figure 69 C is the instantaneous phase change (standard deviation is pointed out) that joins with the spot correlation that lacks cell.The phase place that the color-bar representative is expressed with nm.

Embodiment

The harmonic interference mensuration that is used for range observation:

The present invention relates to intersect with phase place is the system and method for basic measurement optical range, and described system and method is by introducing uneven integer or the 2 π blur level problems of solving of chromatic dispersion in interferometer.The relative height of two points that adjoin is poor on the accurate surface measurements of preferred embodiment energy of described method.In addition, the accuracy that has found that the refractive index of sample only is subjected to the restriction of the precision of experiment measuring sample actual (real) thickness.

In based on the interferometry (HPI) of harmonic phase, replace one of them low-coherence light source to allow to use the CW heterodyne signal that is associated as the form of measuring the optical scale that hangs down coherence's heterodyne signal with continuous wave (CW) light source.Low-coherence light source provides spectral bandwidth, for example, for 1 micron wave length greater than 5nm.One of advantage of using described improved HPI is that the actual measurement phase place is now to length scales nL rather than to (n _{λ 2}-n _{λ 2}) the L sensitivity, wherein n is the refractive index under low relevant wavelength.Quantity n is in fact than synthetic quantity (n _{λ 2}-n _{λ 2}) much useful.By a low relevant wavelength is regulated slightly, for example, about 2nm, people can find that numerical value nL does not have 2 π blur leveles and the sensitivity of Subnano-class is arranged.This method uses CW difference interference signal as the reference field optical scale of measuring optical range.

The system that uses the low-coherence light source that obtains easily to measure the interferometric optical distance has realized the resolution of about tens wavelength.Although this technology is more insensitive, it needn't solve 2 π blur level problems.Preferred embodiment comprises uses the low coherence interferometry of phase place with the optical range of any length of inferior nanometer accuracy measurement.This method uses low coherence's phase place technology that intersects to determine the integer interference fringe and use self-metering additional phase information to obtain the mark striped exactly.In addition, its x-ray tomography section of depth resolution being provided and can being used to the stratification sample is measured.Because described method can accurately be measured long optical range, so it can be used for exactly determining the refractive index of numerous materials.Because this is based on the method for phase place, so the refractive index of finding is the phase potential index of refraction rather than colony's refractive index like this.

Fig. 1 illustrates the preferred embodiment that comprises the system 10 of improved Michelson interferometer of the present invention.Input light wave 12 is by from Ti: sapphire laser (for example, with 775.0nm emission) 150-fs locked mode light wave and the double-colored composite light beam of forming from the light wave of continuous wave (CW) 1550.0nm of for example semiconductor laser.In preferred embodiments, described method is calculated optical range according to CW wavelength (1550.0nm in this embodiment exactly says so), and all optical ranges all are based on this basic calculation.Composite light beam is divided into two at spectroscope 14 places.A part of signal incides on the target sample 16, and another part incides preferably on the benchmark catoptron 32 that the speed with (for example) about 0.5 mm/second moves, and the latter brings out Doppler shift on reference beam 34.Doppler shift can bring out with other device, for example, and by using electrooptic modulator.The light beam of backreflection by combination once more, is divided into their wavelength component by dichroic mirror 18 at spectroscope 14 places, and is opened measurement in 20,22 minutes by photodetector.Consequent signal is by analog to digital converter (ADC) 24 (for example, the A/D converter of 16 100kHz) digitizing.Data processor such as personal computer (PC) 26 is communicated by letter with ADC24, so that further deal with data.Consequent heterodyne signal has the passband around their center heterodyne frequencies separately and is carried out the Hilbert conversion so that infer the phase place of heterodyne signal correspondence, ψ under their doppler shifted frequencies separately _CWAnd ψ _LCSubscript CW and LC represent the continuous wave component of 1550.0nm and low coherence's wavelength component of 775.0nm respectively.

Then, the centre wavelength of low coherence's light wave is regulated about 1-2nm, and measure second group of ψ _CWAnd ψ _LCNumerical value.According to this two group number-reading, can make various interface localization in the target sample with inferior nano-precision.The data processing that is used for localization is described hereinafter.

Consideration is by distance spectroscope 14 unknown distance x ₁The sample formed of single interface.All is known quantity at each time point apart from x from spectroscope 14 to benchmark catoptron 32 the scanning of benchmark catoptron.

Seek x ₁The method of approximate value be by scanning x in the low coherence's light beam that reconfigures and the consequent heterodyne signal of monitoring.When x is approximately equal to x ₁The time, the peak value of heterodyne signal amplitude is expected.The degree of accuracy of this method is subjected to the coherent length L of light source _CSignal to noise ratio (S/N ratio) quality limitations with heterodyne signal.Under the experiment condition of reality, the x that determines ₁Error can not be better than 1/5th of coherent length.

Nominally supposing the coherent length of typical low-coherence light source is about 10 μ m.The error that this means the method for described definite length is limited to about 2 μ m.

When considering the phase place of heterodyne signal, the different components of detected heterodyne signal can be expressed as:

$I_{\mathrm{heterodyne}}=E_{\mathrm{ref}}{e}^{i(2\mathrm{kx}-\mathrm{\ωt})}{E}_{\mathrm{sig}}{e}^{-i(2k{x}_1-\mathrm{\ωt})}+c.c.$

$=2E_{\mathrm{ref}}{E}_{\mathrm{sig}}\cos \left(2k(x-x_1)\right)---\left(1\right)$

E wherein _REfAnd E _SigBe respectively benchmark electromagnetic field amplitude and signal electromagnet field amplitude, k is the optics wave number, and ω is an optical frequency.This path of twice process of the factor 2 expression light waves in the index promptly shines catoptron/sample and returns spectroscope.

Note that when x accurately with x ₁When matching, heterodyne signal is supposed to reach peak value.Two light beams that return are among the constructive interference.So this character is used to the localization interface.x ₁Be that x numerical value when seeking two light beams and be in constructive interference finds.Because the phase potential energy is measured exactly, so this method provides the length sensitivity of about 5nm.Unfortunately, this method is to need to strengthen calculating, because there are a plurality of heterodyne signals to reach the x value of peak value; In particular, heterodyne signal reaches peak value when x satisfies following formula:

x＝x ₁+aλ/2 (2)

Wherein a is an integer, and λ is an optical wavelength.This is the performance of 2 π blur level problems.

Embodiment preferred comprises the method at the peak of distinguishing correct.Note that working as x equals x definitely ₁The time, no matter optical wavelength, heterodyne signal reaches peak value.On the other hand, as be illustrated in fig. 2, peak afterwards depends on wavelength.Fig. 2 illustrates the low coherence's heterodyne signal that is associated with reflecting interface 52 in the sample.So, by regulating low coherence's wavelength, this heterodyne signal be compressed in the interface and with can distinguish x-x definitely ₁The correct peak that is associated of situation around.It should be noted that heterodyne signal may be compressed at the interface or expand, and depends on the adjusting direction on every side.The direct-vision method of visual inspection localization is to draw to get into x and equal x definitely ₁Striped or equal x definitely away from x ₁The heterodyne signal of striped expansion.

Owing to following two reasons need the CW light source in described localization method.The first, in fact identification this numerical value in absolutely accurate ground is very difficult in interferometer.The CW component of interferometer allows in scanning benchmark catoptron, very accurately measurement of x.In specific preferred embodiment, in order to determine in the sample distance between two interfaces, number goes out to occur in x ₁Equal the position and the x of the distance at first interface shown in Figure 1 ₂(x ₂=x ₁+ nL, wherein n is the refractive index of sample) equal the number of the CW interference fringe between the position of distance of second contact surface.Fig. 3 illustrate with sample in two heterodyne signals that reflecting interface is associated.Regulate low coherence's wavelength heterodyne signal 78,80 is compressed 82,84 around the interface.

The second, if the phase deviation that is associated with reflection process is arranged, previously described interface localization method may partly be failed.For example, if the surface is a metal, phase deviation is not to be inappreciable so, and equals x definitely as x ₁The time heterodyne signal phase place have certain other numerical value.Although previous method allows at x=x ₁In the correct interference fringe of identification, yet sub-wavelength sensitivity may be traded off.The appearance of CW heterodyne signal allows to find the difference phase place by the HPI method.Understanding to this numerical value allows to make the interface localization with high-caliber sensitivity.

The principle of HPI method can be by exemplary, and refractive index is n under the wavelength of 775nm _775nm, thickness is that the embodiment of L sample illustrates.Two of this sample are being respectively x apart from spectroscopical optical range at the interface ₁And x ₂(x wherein ₂=x ₁+ n _775nmL) position.Note that if optical range at interval greater than coherent length, for example, typically between 1 micron and 100 microns of low-coherence light source, this method could be worked.Otherwise, the interface localization that the heterodyne phase signal that is associated with the interface combines and leads to errors.In order to get across, the back is postponed till in the combination of the phase deviation that is associated with reflection.

Fig. 4 is the scanning that illustrates mathematical description.This scanning is the sample that two interfaces are arranged.Signal 100 is low coherence's heterodyne signals.Vestige 102 is ψ _CW(x).Zoomed-in view 104 is showed the phase place striped.Each striped is corresponding to λ _CWOptical range.Lower ψ _D(x) vestige is two different Δ values.Arrow 106,110 is pointed out phase crossover.Z-axis is unit with the radian.In the time of scanning benchmark catoptron, the phase place of low coherence's heterodyne signal provides with following formula:

${\mathrm{\ψ}}_{\mathrm{LC}}\left(x\right)$

$={\mathrm{<figure-callout\; id="2"\; label="mod"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mod</figure-callout>}}_{2\mathrm{\&pi;}}\left(\arg (R_{\mathrm{LC},1}{e}^{i2k_{\mathrm{LC}}(x-x_1)}{e}^{-{\left(2a(x-x_1)\right)}^2}+R_{\mathrm{LC},2}{e}^{i2k_{\mathrm{LC}}(x-x_2)}{e}^{-{\left(2a(x-x_2)\right)}^2})\right)$

$\&ap;h_c(x-x_1)\mathrm{<figure-callout\; id="2"\; label="mo\; d"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mo</figure-callout>}{d}_{}{}_{2\mathrm{\&pi;}}\left({2k}_{\mathrm{LC}}(x-x_1)\right)+h_c(x-x_2)\mathrm{<figure-callout\; id="2"\; label="mo\; d"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mo</figure-callout>}{d}_{}{}_{2\mathrm{\&pi;}}\left(2k_{\mathrm{LC}}(x-x_2)\right),---\left(3\right)$

R wherein _LCjBe the reflectivity of interface j under low coherence's wavelength, k is the optics wave number, a=4ln (2)/l _c, l _cBe coherent length, x is that the benchmark catoptron is to spectroscopical distance, h _c(x) be sectional-continuous function, | x|＜2l _cThe time, its numerical value is 1, otherwise is 0.The factor 2 in the index is owing to light path in retroeflection geometry is doubled effectively.Formula 3 reflection since noise not energy measurement considerably beyond this fact of phase place of coherence's envelope.Though setting up coherence's envelope of model is Gaussian on profile, same Phase Processing all is effective for the profile of the envelope of any slow variation.

The phase place of CW heterodyne signal provides with following formula:

${\mathrm{\&psi;}}_{\mathrm{cw}}\left(x\right)={\mathrm{<figure-callout\; id="2"\; label="mod"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mod</figure-callout>}}_{2\mathrm{\&pi;}}\left(\arg (R_{\mathrm{cw},1}{e}^{i2k_{\mathrm{cw}}(x-x_1)}+R_{\mathrm{cw},2}{e}^{i2k_{\mathrm{cw}}(x-({\mathrm{<figure-callout\; id="1"\; label="x"\; filenames="A20048002083801151.png,A20048002083801161.png"\; state="\{\{state\}\}">x</figure-callout>}}_1+n_{1550nm}L)})\right)$

$=\mathrm{<figure-callout\; id="2"\; label="mo\; d"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mo</figure-callout>}{d}_{}{}_{2\mathrm{\&pi;}}\left(\arg \left(\stackrel{\&OverBar;}{R}{e}^{i2k_{\mathrm{cw}}(x-\stackrel{\&OverBar;}{x})}\right)\right)=\mathrm{<figure-callout\; id="2"\; label="mo\; d"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mo</figure-callout>}{d}_{}{}_{2\mathrm{\&pi;}}\left(2k_{\mathrm{cw}}(x-\stackrel{\&OverBar;}{x})\right),$ (4)

R wherein _CWjBe the reflectivity of interface j under the CW wavelength, n _1550nmBe the refractive index of sample, R and X are respectively effective average reflectances and arrive spectroscopical effective mean distance.If two light source center wavelength are selected like this, consequently

k _LC＝2k _CW+Δ，(5)

Wherein Δ is the little additional skew of having a mind to, the poor phase place ψ of so this form _DObtained:

ψ _D(x)＝ψ _LC(x)-2ψ _cw(x)

＝h _c(x-x ₁)mod _2π(4k _cw( x-x ₁)+2Δ(x-x ₁)) (6)

+h _c(x-x ₂)mod _2π(4k _cw( x-x ₂)+2Δ(x-x ₂))

Above-mentioned amount is provided at (x at interval ₂-x ₁) in striped the cardinal principle number and the mark striped of sub-wavelength precision is provided.

When the parameter Δ is changed on a small quantity (corresponding to the approximately wavelength shift of 1-2nm), ψ _D(x) slope is around x=x ₁And x=x ₂Point rotate.In other words, in different Δ values and the crossing place scanning phase place of described point.From x ₁To x ₂Optical range can be by calculating ψ _CW(x) striped of process is found out between two phase crossovers.The twice of the quantity that so finds non-integer S _FringePoint out, and corresponding to the striped number under the low relevant wavelength.With regard to single interface,, can find out by carrying out various scanning with a plurality of additional Δ values with the corresponding point of interface location if a plurality of phase crossovers occur.Interface location is unique position, at this position ψ _D(x) all will intersect for all Δ values.

Phase-shift information is used for further making interface spacing localization.In particular, at x=x ₁And x=x ₂Difference between the phase deviation at place is:

$S_{\mathrm{phase}}=\frac{{\mathrm{<figure-callout\; id="2"\; label="mod"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mod</figure-callout>}}_{2\mathrm{\&pi;}}({\mathrm{\&psi;}}_D(x=x_1)-{\mathrm{\&psi;}}_D(x=x_2))}{2\mathrm{\&pi;}}=\frac{{\mathrm{<figure-callout\; id="2"\; label="mod"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mod</figure-callout>}}_{2\mathrm{\&pi;}}\left(4k_{\mathrm{cw}}(x_2-x_1)\right)}{2\mathrm{\&pi;}}\&CenterDot;---\left(7\right)$

This uses high-sensitivity measurement mark striped.

Absolute optical interval (x ₂-x ₁) can be by following formula accurately from S _FringeAnd S _PhaseDetermine:

${(x_2-x_1)}_{\mathrm{measured}}={\left({\mathrm{<figure-callout\; id="775"\; label="n"\; filenames="A20048002083800961.png"\; state="\{\{state\}\}">n</figure-callout>}}_{775\mathrm{nm}}L\right)}_{\mathrm{measured}}=$

$\frac{{\mathrm{\&lambda;}}_{\mathrm{cw}}}{4}\left(\right[\mathrm{int}\left(S_{\mathrm{fringe}}\right)+U(\mathrm{\&Delta;S}-\frac{1}{2})-U(-\mathrm{\&Delta;S}-\frac{1}{2})]+S_{\mathrm{phase}})$ (8)

Δ S=rEs (S wherein _Fringe)-S _Phase, U () is the unit step function.Here, int () and res () represent the integral part and the mark part of independent variable respectively.First is passed through S _PhaseAnd S _FringeFractional part between error reduce to minimum with the correct integer number of localization optical range to striped.The optical interval error at measurment only is subjected to S _PhaseMeasuring error restriction.In embodiments, such error is transformed at the about (n of 0.5nm _775nmL) _MeasuedIn error.S _PhaseMeasuring error only need be less than half striped, so that correct interference fringe can be established; Satisfied this criterion, it does not enter (n _775nmL) _MeasuedError.Maximum measurable optical range only depend on system exactly number go out the fringe number purpose ability between two intersection points and the frequency stability of light source.

Above-mentioned formula is the concise expression that is used for finding the method for correct striped and mark striped.Operation can and be showed by selection by the following examples to be made based on S _PhaseAnd S _FringeBetween the numerical value of evaluated error minimum determine that correct Fig. 5 that estimates illustrates.Suppose S _FringeAnd S _PhaseBe 26.7 and 0.111.From S _PhaseMeasurement result, the numerical value of optical range is:

(n _775nmL) _measued＝λ _CW(a+0.111)/4 (9)

Wherein a is an integer.Provide S _FringeNumerical value, possible (n _775nmL) _MeasuedNumerical definiteness is at following three numerical value: λ _CW(25.111)/4, λ _CWAnd λ (26.111)/4 _CW(27.111)/4.If λ _CW(27.111)/4 numerical value is near λ _CW(S _Fringe)/4, it is exactly (n so _775nmL) _MeasuedCorrect estimation.

Be used for the preferred embodiment that the harmonic relationships light source is measured for the interferometry on basis, suitably Xuan Ding light source is to allowing to minimize and preferably eliminate and otherwise make the high-precision optical range observation become impossible flutter effect in the interferometer with the go on business method of phase place of deduction.Eliminate shake and also allow to compare the scanning of finishing at different time.

For the ability of the preferred embodiment that proves this method, described system is used to seek and visit the top surface of vitreosil cover glass of actual (real) thickness L=237 ± 3 μ m and the optical range between the lower surface.In this embodiment, have the π phase deviation that is associated with reflection from first interface, this indicates the transition of positive refraction index.Therefore, in formula 1 and 2, have and factor R _{LC, 1}And R _{Cw, 1}The e that is associated ^{-i π}.This causes about S _FringeAnd S _Phase1/2nd correction factor.Fig. 4 is illustrated in exemplary scanning result under the LC wavelength of 773.0nm and 777.0nm.Four groups of scanning results are summarised in the (n of expression about the quartz cover slide _775nmIn the table 1 of measurement result L).The repeatability of experimental data shows that light source is sufficiently stable aspect frequency.

Table 1

	λ CWS fringe/4(μm)	λ CWS Phase/4(μm)	(n 775nmL) measued(μm)
				Group 1	350.86±0.17	0.3496±0.0004	351.0371±0.0004
Group 2	351.08±0.17	0.3497±0.0004	351.0372±0.0004
				Group 3	351.15±0.16	0.3502±0.0004	351.0377±0.0004
Group 4	351.04±0.18	0.3498±0.0004	351.0373±0.0004
				On average			351.0373±0.0004

Experimental data produces the absolute optics range measurements that inferior nano-precision is arranged.The optical range of being found is associated with low-coherence light source.The CW heterodyne signal serves as optical scale.If the L of quartz cover slide is accurately known, so quartzy n under the 775.0nm wavelength _775nmJust can be from (n _775nmL) _MeasuedDetermine with very high accuracy.

As an alternative, do not know the accurate numerical value of L, determine at the optical range of low-coherent light ripple under these wavelength and the CW light wave measurement correspondence under their harmonic waves separately by using in refractive index specific energy under two different wavelength.Use a series of low coherence's wavelength, the dispersion profile figure of material can be determined exactly.Dispersion profile figure plots figure to the index difference under the various different wave lengths.According to embodiment preferred, these experimental results predict that the degree of accuracy of about seven position effective digitals can realize with the sample of about 1 millimeters thick.

In a further preferred embodiment, the light source of described system changes into low coherence's superluminescent diode (SLD) of 1550.0nm emission and the CWTi that launches with 775.0nm: sapphire laser.By regulating the working current by SLD, centre wavelength is changed about 2nm; This is fit to realize that phase place intersects.Use the described preferred embodiment of system of the present invention, optical range can be measured at 1550.0nm.Adopt the ratio of described measurement result and the measurement result of front, quartzy refractive index compares n _775nm/ n _1550nmCan be determined.It should be noted that the refractive index ratio of being found is owing to used light source in described preferred embodiment is suitable for the harmonic relationships wavelength.Other light source measurement of suitably selecting of the refractive index specific energy of other wavelength.For relatively, the data of glass and acrylic plastics correspondence are listed in the table 2 n as different materials _775nm/ n _1550nmMeasurement result.

Table 2

	n 775nm/n 1550nm
		Quartzy	1.002742±0.000003
Glass (German Borosilicate)	1.008755±0.000005
		Acrylic plastics	1.061448±0.000005

Note that when low coherence's wavelength when being half of CW wavelength some used formula be different from the formula that previously herein occurs slightly.For example:

${\mathrm{\&psi;}}_{\mathrm{LC}}\left(x\right)$

$={\mathrm{<figure-callout\; id="2"\; label="mod"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mod</figure-callout>}}_{2\mathrm{\&pi;}}\left(\arg (R_{\mathrm{LC},1}{e}^{i2k_{\mathrm{LC}}(x-x_1)}{e}^{-{\left(\frac{2}{l_c}(x-x_1)\right)}^2}+R_{\mathrm{LC},2}{e}^{i2k_{\mathrm{LC}}(x-x_2)}{e}^{-{\left(\frac{2}{l_c}(x-x_2)\right)}^2})\right)$

$\&ap;h_c(x-x_1){\mathrm{<figure-callout\; id="2"\; label="mod"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mod</figure-callout>}}_{2\mathrm{\&pi;}}\left({2k}_{\mathrm{LC}}(x-x_1)\right)+h_c(x-x_2){\mathrm{<figure-callout\; id="2"\; label="mod"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mod</figure-callout>}}_{2\mathrm{\&pi;}}\left({2k}_{\mathrm{LC}}(x-x_2)\right),---\left(10\right)$

${\mathrm{\&psi;}}_{\mathrm{cw}}\left(x\right)={\mathrm{<figure-callout\; id="2"\; label="mod"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mod</figure-callout>}}_{2\mathrm{\&pi;}}\left(\arg (R_{\mathrm{cw},1}{e}^{i2k_{\mathrm{cw}}(x-x_1)}+R_{\mathrm{cw},2}{e}^{i2k_{\mathrm{cw}}(x-({\mathrm{<figure-callout\; id="1"\; label="x"\; filenames="A20048002083801151.png,A20048002083801161.png"\; state="\{\{state\}\}">x</figure-callout>}}_1+n_{1550nm}L)})\right)$

$=\mathrm{<figure-callout\; id="2"\; label="mo\; d"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mo</figure-callout>}{d}_{}{}_{2\mathrm{\&pi;}}\left(\arg \left(\stackrel{\&OverBar;}{R}{e}^{i2k_{\mathrm{cw}}(x-\stackrel{\&OverBar;}{x})}\right)\right)=\mathrm{<figure-callout\; id="2"\; label="mo\; d"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mo</figure-callout>}{d}_{}{}_{2\mathrm{\&pi;}}\left(2k_{\mathrm{cw}}(x-\stackrel{\&OverBar;}{x})\right),---\left(11\right)$

2k _LC＝k _CW+Δ (12)

ψ _D(x)＝2ψ _LC(x)-ψ _cw(x)

＝h _c(x-x ₁)mod _2π(4k _LC( x-x ₁)+2Δ(x-x ₁)) (13)

+h _c(x-x ₂)mod _2π(4k _LC( x-x ₂)+2Δ(x-x ₂))

$S_{\mathrm{phase}}=\frac{{\mathrm{<figure-callout\; id="2"\; label="mod"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mod</figure-callout>}}_{2\mathrm{\&pi;}}({\mathrm{\&psi;}}_D(x=x_1)-{\mathrm{\&psi;}}_D(x=x_2))}{2\mathrm{\&pi;}}=\frac{{\mathrm{<figure-callout\; id="2"\; label="mod"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">mod</figure-callout>}}_{2\mathrm{\&pi;}}\left(4k_{\mathrm{LC}}(x_2-x_1)\right)}{2\mathrm{\&pi;}}\&CenterDot;---\left(14\right)$

${(x_2-x_1)}_{\mathrm{measured}}={\left({\mathrm{<figure-callout\; id="775"\; label="n"\; filenames="A20048002083800961.png"\; state="\{\{state\}\}">n</figure-callout>}}_{775\mathrm{nm}}L\right)}_{\mathrm{measured}}$

$=\frac{{\mathrm{\&lambda;}}_{\mathrm{LC}}}{4}\left(\right[\mathrm{int}\left(S_{\mathrm{fringe}}\right)+U(\mathrm{\&Delta;S}-\frac{1}{2})-U(-\mathrm{\&Delta;S}-\frac{1}{2})]+S_{\mathrm{phase}})---\left(15\right)$

Preferred embodiment with the method that solves 2 π blur leveles is very useful in the application such as the high precision refraction index of range finding of the high precision degree of depth and film solid-state material is determined.

The use of method for optimizing can be by considering that glass plate illustrates.The system that some from the system to glass plate distances at average center of measuring with considerable accuracy are arranged.The system that also has some glass surface degree of roughness that can measure with considerable accuracy.The preferred embodiment of system of the present invention is with the thickness of nanometer sensitivity measure glass plate end face.

The step of implementing the preferred embodiment of the method be used for determining optical range is illustrational with the flow process Figure 124 among Fig. 6 A and the 6B.Method 124 is included in uses two harmonic relationships light sources in the Michelson interferometer, one of them is the CW light source, and another is a low-coherence light source.The sample that need measure the optical range between its interface all is used as the end reflector of interferometer signal arm whenever step 126.Benchmark catoptron in the interferometer reference arm is whenever step 128 all is scanned.This method comprises the step 130 that the reflection merging from signal arm and reference arm is separated according to wavelength then.And then, survey the heterodyne vibration of combined light intensity of wave whenever step 132.Then, whenever step 134 by, for example, Hilbert conversion or any phase place infer that alternative method finds out the phase place of the heterodyne signal of two kinds of wavelength.All deduct the poor phase place of the whole scanning of phase estimation of longer wavelength for twice by phase place from shorter wavelength whenever step 136.Whenever step 137 is all used by the optical wavelength multiple scanning of off resonance slightly.Then, repeating step 130-136.

Then, whenever step 138 all from two poor phrase overlaps of twice scanning discovery on the curve map of the displacement of representing the benchmark catoptron with the x axle.It should be noted that inferring also of difference phase place can be finished with suitable light source or color filter or to the software/hardware signal Processing of single scanning.

Following step comprises in method 124: whenever 140 steps determine that on curve map phase crossover is so that mark the position of example interface.Whenever step 142, by calculating the number of times that the heterodyne signal be associated with the CW light wave stacks with 2 π between two intersection points, determine optical interval between the interface with the accuracy of the mark (for example, about 0.2) that approximately reaches wavelength.By measuring poor phase place at intersection point, further make this spacing localization and/or be refined to the very little mark of wavelength, for example, about 0.001.

In illustrational another preferred embodiment of Fig. 7 of the synoptic diagram that is used as the system of measuring optical range, low-coherence light source may be enough wide aspect bandwidth, for example, surpasses 4nm.When surveying end, be that two detectors 166,176 increase the 3rd detector 174.This causes low coherence's lightwave signal 168 further to be divided into two.Before arriving detector, two light beams are by different wave filters 170,172.Different parts in these two wave filter transmission spectrums.A frequency spectrum component that allows wavelength grow passes through, and second frequency spectrum component that allows wavelength lack passes through.Preferably two transmitted light beams are being separated more than the 2nm aspect their spectrum.

Then, light beam incides on the detector, and their heterodyne signal is handled in the mode that reference Fig. 1 discussed.Advantage according to the method for other possible preferred embodiment is the repetition of the program of eliminating through the low coherence's wavelength that overregulates of this method.These two signals are obtaining in once scanning.

Fig. 8 A and 8B illustrate the flow process Figure 184 that measures the alternative method of optical range according to the preferred embodiments of the invention.Method 184 is included in uses two harmonic relationships light sources in the interferometer, one of them is the CW light source, and another is a low-coherence light source.Whenever step 186, the sample that need measure optical range all is used as the end reflector of interferometer signal arm.Whenever step 188, the benchmark catoptron in the interferometer reference arm all is scanned.This method further comprises whenever step 190 merges the reflection from signal arm and reference arm and according to wavelength they being separated then.Whenever step 192, use wave filter that low coherence's wavelength is further separated.Method 184 comprises the step 194 of surveying the heterodyne vibration with at least three detectors.Next procedure 196 comprises the heterodyne vibration of surveying the combined light intensity of wave.Then, whenever step 198 all by, for example, Hilbert conversion or any phase place infer that alternative method finds out the phase place of the heterodyne signal of two wavelength.Then, whenever step 200, all calculate the poor phase place of each low coherence's signal and CW signal.

Then, whenever step 202, all in curve map, on the x-axle of the displacement of expression benchmark catoptron two poor phase places are overlapped each other.Remaining step 204,206,208 is similar to the step of discussing with reference to Fig. 6 B 140,142,144.

The preferred embodiment of this method definitely can be used for the long arbitrarily optical range of inferior nanometer accuracy measurement.The preferred embodiment of this system can be based on free space or based on optical fiber.It is the preferred embodiment of the system of basic measurement optical range that Fig. 9 illustrates with optical fiber.

Input light wave 256 is included in the low coherence's light wave (wavelength X of the approximate harmonic relationships of propagating in the optical fiber 251 ₁) and CW light beam (wavelength X ₂).Composite light beam is divided into two, and a part of signal incides on object lens 254 and the sample 256 and transmission in optical fiber 253, and another part signal incides on the benchmark catoptron 266 via lens 268 and transmission in optical fiber 251.Doppler shift is introduced in the motion of benchmark catoptron on folded light beam.Folded light beam is merged once more, then, is divided into their composition wavelength component by dichroic mirror 258.Described wavelength component was opened measurement in 260,262 minutes by photodetector.These heterodyne signals that produce under their doppler shifted frequencies separately have the passband around they center heterodyne frequencies separately, and the Hilbert conversion that is done, so that infer the phase place ψ of heterodyne signal correspondence _CWAnd ψ _LC

The method of preferred embodiment can be used for carrying out accurate optical distance measurement.According to such measurement, the optical mass-energy of target object is accurately measured.By the dispersion profile figure of measurement target, the structural property of target and/or chemical mass-energy are calculated.At biomedical sector, the preferred embodiments of the invention can be used for accurately determining with noncontact and non-invasive mode the chromatic dispersion character of biological organization.Such dispersion measurement can be used on eye's cornea or the aqueous humor.The sensitivity that is realized is enough to survey the optical change fixed according to concentration of glucose.In the preferred embodiment of the inventive method, blood sugar level can be determined by the aqueous humor of non-intrusion measurement eyes or the dispersion profile figure of glass metal or cornea.

As discussed above, can measure optical range very delicately based on the interferometry of phase place.Yet described mensuration is subjected to the restriction of well-known problem in this field of 2 π blur leveles and so on for example in their application facet.The crux of this problem is and impossible separates the length of 10.1 wavelength and the length field of 11.1 wavelength.The preferred embodiments of the invention have overcome this restriction and have allowed the absolute optical distance measurement of Ya Nami accuracy.

Many methods based on phase place that change with the sensitivity measure optical range of approximate nanometer range are arranged.As long as this variation is very little and be progressive, described variation just can be followed the tracks of continuously.Exist to measure the low coherent approach of absolute optical range, described method arrives the delay of detector of light wave that is approximately several microns different boundary reflection from catoptron sensitivity and measures absolute optical range by following the tracks of.As discussed above, in interferometer, use method to create conditions in CW light source and the low-coherence light source for measuring optical range.The heterodyne phase of the signal that is associated with two kinds of wavelength is relevant in essence.By handling the phase place of each preferred embodiment, the motion noise is reduced to minimum and preferably eliminates from our measurement.

The application of preferred embodiment is to use the measurement result of the refractive index of the vitreous humor of eyes and/or aqueous humor to determine glucose level.The sensitivity of this technology provides the ability with appropriate clinically sensitivity measure chemical concentrations.One of apparent in view application of the method for preferred embodiment is to determine blood sugar level by the measurement of finishing on eyes.The glucose level of the fluid glucose level of inessential clinically time delay reflection blood in the eyes.

The vitreous humor in illustrational at least two the set of wavelengths measurement eyes that separate of method use Figure 10 of preferred embodiment and/or the optical path length of water sample liquid layer.This method is measured the refractive index under low coherence's wavelength and the product of the actual pitch between two interfaces.Wavelength by changing low-coherence light source (and suitably change CW wavelength in order to mate) is with different wavelength measurement index difference.For example, measuring for one group is to finish with the low-coherence light source of tunable 500nm and 1 micron CW light source, so that extract n _500nmL, wherein L is vitreous humor and/or the aqueous humor actual (real) thickness at measurement point.It is to finish with the low-coherence light source of tunable 1000nm and the CW light source of 1800nm that another group is measured, so that extract n _900nmL.By obtaining the ratio of these two measurement results, the refractive index of vitreous humor and/or aqueous humor compares n _500nm/ n _900nmInferred.Adopt existing sensitivity, for example, the sensitivity of 0.5nm light path, the preferred embodiment of this system can be 10 with the mankind's the vitreous humor and/or the Materials Measurement sensitivity of aqueous humor at thickness ^-8Refractive index compare n _500nm/ n _900nmThis this sensitivity offers about 0.25 milligram/deciliter glucose level and changes.Suppose that typical blood sugar level is about 100 milligrams/deciliter, the preferred embodiments of the invention are fit to the blood sugar chemical examination very much.The selection of optical wavelength is flexibly, more than just explanation for example of the wavelength of Shi Yonging.With regard to the highest sensitivity, the wavelength interval is preferably big as far as possible.Preferred embodiment comprises the interval greater than 500nm.

Be not enough to determine that than other chemicals that is changing owing to appearance in vitreous humor and/or aqueous humor under the situation of absolute blood sugar level, a series of more completely optical path lengths are measured and can be carried out in such refractive index under a series of other wavelength.This group measurement result more completely allows by making measurement result and the known glucose and the dispersion profile figure match of other chemicals determine gentle other the chemical concentration of G/W.

The preferred embodiments of the invention can be applied to semiconductor manufacturing industry as measuring technique.Because the preferred embodiment of described method is untouchable with nondestructive, so it can be used for the thickness of in manufacture process monitoring semiconductor structure.In addition, the composition of semiconductor structure can be to chemically examine with the mode of the as much of the characteristic discussion of measuring with regard to vitreous humor and/or aqueous humor.

Phase measurement and imaging system:

Other possible preferred embodiment of the present invention relates to light wave makes little biosome or characteristic imaging.These embodiments can be applied to numerous areas, for example, and stechiology and Neuscience.These preferred embodiments are based on the principle of phase measurement and imaging technique.Use the science motivation of phase measurement and imaging technique to originate from, for example, can comprise the cell biology of the pattern of sub-micron level of the imaging cause that dysplasia, cell communication, neuron transmission and genetic code are carried out ad lib.The structure of subcellular fraction component and dynamics can not use existing method and technology (comprise, for example, the scattering of X ray and neutron) to study with their state of nature now.Otherwise, the cell machine can be studied with its state of nature with the technology of the nanometer resolution on light wave basis.Therefore, the preferred embodiments of the invention comprise based on the system of the principle of interferometry and/or phase measurement and are used to study stechiology.These systems comprise the low coherence's interferometry (LCI) of using the optical interdferometer Measurement Phase or the principle of wherein using the light wave scattering spectroscopy (LSS) of the interference within the cell component itself, or the principle of LCI and LSS can merge in system of the present invention in replacement scheme.

The preferred embodiment that is used for phase measurement and imaging system comprises initiatively stable interferometer, isolates intervention instrument, altogether to the light path interferometer with the interferometer of variate is provided.The embodiment that relates to differential measuring system comprises 2 heterodyne ineterferometers and double beam interferometer.Use the phasecontrast microscope that can comprise the modulation of usage space light wave altogether to the embodiment of light path interferometer.

The low coherence's interferometry (LCI) of optics is finding many application aspect the vectorial research of biology.The most widely used LCI technology is to make the 2D of biological sample or the optical coherence tomography art (OCT) of 3D backscattering profile imaging.Drexder, W. wait the people " In vivo ultrahigh-resolution optical Coherencetomography " (Optics Letters, Volume 24, No.17, pages 1221-1223) described the LCI technology in, by quoting as proof its whole instructions were incorporated at this.OCT has the degree of depth sensitivity of the coherent length restriction that is subjected to used light source.Ultra broadband light source can be differentiated about 1 micron size characteristic.

The low coherence's interferometry of phase sensitivity is responsive to the variation of sample sub-wavelength light path.The main difficulty of phase sensitivity LCI is the phase noise that the light path change causes in the interferometer both arms.Can be used for the stellar interferometer phase noise by the different wavelength of laser bundle of same light path almost, then described phase noise be deducted from the sample signal that similar noise is arranged, so that extract real sample phase deviation.Other researcher has used along the differential phase contrast or the birefringence of measuring high phse sensitivity altogether to the laser polarization of light path quadrature.In these two kinds of technology, all scan the reference arm path, and need COMPUTER CALCULATION, so that infer phase place from consequent striped data (via the Hilbert conversion); In addition, the necessary 2 π blur leveles of using in the phase-unwrapping algorithm elimination phase measurement.Strip-scanning and message processing program reduce measuring speed in fact and may increase noise.

The system that comprises initiatively stable interferometer:

The preferred embodiments of the invention are used the LCI method, and wherein interferometer is surveyed very little phase deviation by stable permission of the active of reference beam incessantly with high bandwidth and minimum Computer Processing.The reference beam that is locked in the arbitrary phase angle is not having to provide direct sample phase measurement under the reference arm scan condition.Embodiment preferred provides the two and three dimensions phase imaging.

It is stable by the active of reference laser beam interferes that embodiment preferred relies on the Michelson interferometer.Initiatively the synoptic diagram of the preferred embodiment of stable interferometer 300 is illustrated among Figure 11.Initiatively stable Michelson interferometer system 300 comprises catoptron 306; Mobile mirror 310; Spectroscope 304; Phase-modulator 308; Detector 318; Local oscillations source 320,322; Frequency mixer 316 and summing amplifier 312.The continuous-wave laser beam that is separated by spectroscope 304 crosses two interferometer arm and is reconfigured at detector 318 places.An arm of interferometer comprises phase modulation component 308, for example electrooptic modulator or be installed in catoptron on the piezoelectric transducer.Regulating significantly and can finish optical path difference by translation catoptron 310 or any other variable optical delay line.Treating apparatus (for example, computing machine 315) be used to provide feedback and communicate by letter with the electron device of handling the phase deviation measurement result.Electronic image display 317 is used for showing phase deviation and relevant image.

Phase differential between two interferometer arm is modulated by sinusoidal curve:

φ＝ψ+ψ _dsin(Ωt) (16)

ψ=k (L wherein ₁-L ₂)=k Δ L is two phase differential between the arm, ψ _d＜2 π are depth of modulation, and Ω is a modulating frequency.The interferometer signal that is detected is to be used for the relevant addition of light beam of two arms of self-interference instrument to provide:

I＝I ₁+I ₂+2(I ₁I ₂) ^1/2cosφ (17)

The signal that nonlinear relationship between I and the φ causes being detected has the component under many harmonic frequencies of modulating frequency Ω.First (the I ^Ω) and the second (I ^{2 Ω}) harmonic term provides with following formula:

I ^Ω＝4J ₁(ψ _d)(I ₁I ₂) ^1/2sinψsin(Ωt) (18)

I ^2Ω＝4J ₂(ψ _d)(I ₁I ₂) ^1/2cosψcos(2Ωt) (19)

I ^ΩAnd I ^{2 Ω}Finish by frequency mixer 316 or lock-in amplifier by Ω and 2 Ω demodulation respectively, and two signals all are exaggerated as the function of ψ, so that provide equal amplitude:

V ₁＝V ₀sinψ (20)

V ₂＝V ₀cosψ (21)

Use the analog or digital circuit, linear combination V ₀Be to calculate with time dependent parameter θ:

V _θ＝cosθ*V ₁-sinθ*V ₂＝V ₀sin(ψ-θ) (22)

This signal is used as any zero crossing that error signal is locked in interferometer positive slope.V _θ(t) be fed back to that phase-modulator (high frequency) and path length modulator (low frequency) are integrated before, filtering and amplification be so that initiatively eliminate the interferometer noise.Linear combination V _θ(t) be used as error signal so that allow to lock onto arbitrary phase θ.

Stable interferometer can combine with the low coherence's interferometry of phase sensitivity as described here.All can realize at this system component that is described by free space optical system or fiber optic system.For clear, illustration is showed the realization of free space optical system.

The schematic diagram shows of leaning on the stable interferometer of reference beam that is used for optical delay phase sensitivity LCI is at Figure 12.From the light beam 353 of low-coherence light source in stable interferometer by the path identical with locked beam 355.By changing (stablizing) path length difference between two arms of interferometer, prepare the output beam of forming by two parts of LC light beams " copy " sum of the optical path delay of modulating by interferometer locking modulating frequency that highly stable continuous variable is arranged therebetween.

Sample 382 is placed on the cover glass, this cover glass with side that sample contacts on be coated with the coating of the anti-LC wavelength reflection of last layer.The LC light beam focuses on by cover glass and sample by micro objective 380.The backscattering light wave be collect with same optical system and focus on the detector 366.The signal that is detected is the auto-correlation of backscattering scanning electromagnetic field and time delay Δ L/c.It can be demonstrated, so that be presented at zero-lag and and illustrational several to the interferogram under the corresponding delay of twice optical path length between scattering or the reflecting surface according in sample arm of Figure 13.Specifically, cover glass does not have the side of coating to be positioned at position apart from the about cover-glass thickness d of sample; Be to see by optical path delay nd under the situation of n from the interference signal of sample in the refractive index of glass.

Because Δ L=～2nd, sample signal is pressed modulating frequency by demodulation, the continuous coverage of sampling phase place by frequency mixer or lock-in amplifier.For the zero crossing in the low coherence's signal that locks demodulation, interferometer locking phase θ can change with electronics method successively.In this way, the time of interferometer locking phase progress is used as the direct measurement of sample phase place.Described locking scheme has the advantage of the amplitude that is independent of sample signal.

Described system is similar to twin-beam optical computer tomography (OCT) technology according to preferred embodiment, because optical delay was ready to before low coherence's light wave enters sample, and the signal that is detected is insensitive to the variable in distance between sample and the interferometer.In other possible preferred embodiment, also can use the Mach-Zender interferometer configurations to prepare low coherence's light beam.

By variable attenuator being introduced one or two arm of interferometer, the relative amplitude of the electromagnetic field of two time delays can be modulated in order to optimize interference signal.

Lean on the synoptic diagram of the LCI of the stable phase sensitivity of reference beam to be illustrated among Figure 14 A.This system component and Figure 11 are similar, but replace a catoptron of interferometer with the sample on the cover glass 430.From two light beams the 422, the 424th of two low-coherence light sources (LC1 and LC2), the incident beam on the interferometer input end.Reference beam has and is equivalent to or greater than the coherent length of cover-glass thickness (for example, about 150 microns).The cover glass reflection is used to lock interferometer.The reference beam of short coherent length prevents that the interferometer locking is subjected to the influence from micro objective and other surperficial false reflection.

In order to distinguish the reflection of sample surfaces and rear surface, signal beams has than the little several-fold coherent length of cover-glass thickness.Reference arm length is conditioned for the interferogram that provides from sample, and as previously described, this signal in order to provide the illustrational sample phase place of Figure 15 A and 15B by demodulation.Interferometer causes the sample phase measurement relevant with this interface about the locking that cover glass does not have coated side, and almost gets rid of all outside interferometer noises.

The optical delay method of describing with reference Figure 11 relatively, this preferred embodiment has signal beams and reference beam both shortcomings as the incident beam on the sample.For biomaterial, living cells especially, this perhaps limits the reference beam power that may use, thereby causes reducing lock mass.On the other hand, scanning benchmark catoptron allows to recognize more straight from the shoulder the reflection from sample.Treating apparatus such as computing machine 453 be used to provide feedback and communicate by letter with the electron device of handling the phase deviation measurement result.Electronic image display 455 is used for showing phase deviation and relevant image.

Embodiment preferred can use two kinds of methods to make sample phase imaging on certain zone.In preferred first method, incident beam can be as scanning on sample along the X-Y direction in most of OCT equipment.In the embodiment that comprises the LCI stable, must in beam flying, keep the locking of reference beam interferometer by reference beam.According to second method, charge-coupled device (CCD) or photodiode array can be used for surveying these signals and not need to scan.Figure 16 illustrates the imaging system 500 that is used for stable phase sensitivity LCI.This optical system be used for throwing light on extended zone and make scattered light imaging on detector.Figure 17 illustrates the deployment schemes of the simplification of system configuration, so that illustrate optical design according to the preferred embodiments of the invention.Solid line is represented incident ray, and dotted line is represented backscattering light.

With regard to the CCD imaging, the measurement of relative phase can be finished by the sequence of analyzing 4 width of cloth images, and every width of cloth all is different from the last width of cloth aspect phase place, differ pi/2.Figure 14 B illustrates in the embodiment that is used for hanging down according to the stable phase sensitivity of active that the present invention uses the catoptron of band piezoelectric transducer (PZT) 461 to produce phase deviation the use CCD of the system imaging of coherence's interferometry.Circuit 469 is the electron devices that are used for using PZT to produce the electron device of phase deviation and are used for the detecting phase skew.CCD is the cell array that is integrated among the electronic chip of a slice compactness.The CCD controller 477 that treating apparatus own and such as computing machine 478 is connected is communicated by letter with CCD.Image display 479 is used for showing phase deviation and relevant image.

For the high bandwidth phase imaging, can be from the signal of photodiode array by first harmonic and individually demodulation of second harmonic; This allows to measure clearly the phase place of each pixel.

The higher sensitivity of the preferred embodiment of interferometer measuration system of the present invention and bandwidth provide new possibility for measure little optical phase shift in biological or abiological medium.For example, these preferred embodiments can be studied the motion and the fluctuation of cell membrane.The phase sensitivity LCI of dual wavelength has been used for observing the cell volume adjusting and the film dynamics of human colon cell culture.Recently, in culture, added the low-frequency oscillation that sodium azide is observed cell membrane afterwards.Preferred LCI embodiment allows by less time scale research film dynamics, and hot in this case fluctuation and the mechanical vibration that drive may be prior.Two-dimensional imaging method according to the preferred embodiments of the invention allows research film fluctuation when collecting interactive cell.Vibration and mutual relationship can provide the information of signaling about cell.

The preferred embodiments of the invention can be used for the measurement of neuron action potential.Optical means for the neuronic electric signal of improved non-intrusion type ground monitoring in Neuscience has very big interest.Present method is faced upward by responsive to calcium or to the dyestuff of voltage-sensitive, they have many problems, comprises short, photic toxicity of life-span and response time slowly.

Decades ago people just known action potential in nerve fibre and cell body with optical change.In addition, between stimulation period, neural having shown presents of short duration volume increase.These change according to the phase transition in the cell membrane and since the index offset that causes that redirects of the dipole in the cell membrane obtain explaining.

Phase sensitivity LCI method according to preferred embodiment can be used for measuring optics and the machinery variation that is associated with action potential.The bandwidth that increases allows by the action potential markers of about 1ms Measurement Phase delicately.The long-term measurement and providing that the preferred embodiments of the invention can be used to provide the non-intrusion type of nerve signal makes the ability of many neurons imagings simultaneously.It is important for understanding brain that these embodiments help to analyze the graphic formation of neururgic time and space.Known and action potential accompany, and little (≈ 10 ^-4Rad) fluctuation of index offset and film (can be detected out in＞1kHz) the preferred embodiment in high-level responsive speed and the high bandwidth of providing of the present invention.These embodiments are used the method for eliminating noise, for example, prevent the isolation method that noise enters; Use feedback element to eliminate the stabilization method of noise; Provide to feed back and eliminate noise noise effect is reduced to minimum altogether to the variate of light path interferometry.

Embodiment described here can be used to many medical applications.For example, the cortex mapping can be finished during neurosurgery, and comparing with the electrode method of prior art is making progress aspect speed and the resolution.In addition, these preferred embodiments can be used for making epilepsy focus localization during neurosurgery.These embodiments also can be monitored the retina neural activity in the eyes.The high-speed two and three dimensions scanning that provides owing to these embodiments is provided in other application of the preferred embodiments of the invention; High dynamic range that photodiode detector provides and DC suppress; The nanoscale imaging of cell biology aspect; The sign of epithelial tissue and the detection of vibration of membrane for example, but are not limited to, auditory cell and blood vessel.

The system that comprises double beam interferometer:

The preferred embodiments of the invention comprise the low coherence's interferometer of the optical delay phase sensitivity based on optical fiber that is integrated in traditional light wave microscope.Synchronous electricity and optical measurement can be finished in the culture of hippocampal neuron.Embodiment preferred comprises the imaging system that comprises photodiode array or quick scanning light beam.Being used for the neuronic method of optical stimulation combines with the LCI measurement of action potential and can form the exceedingly useful new tool of other basic problem that is used for studying neural network dynamics, synaptic plasticity and Neuscience aspect.

Another embodiment is applied to brain section with the phase sensitivity imaging technique, even the live body neuron.Follow the tracks of and the motion on compensation brain surface be relevant challenge.The optical scattering restriction may be extracted the degree of depth of neuron signal, but about 100 microns degree of depth may be possible.

The preferred embodiment of the stable interferometer of the active of Miao Shuing has comprised two wavelength systems before this, and wherein first wavelength is used to stable and second wavelength is used to phase measurement.Figure 18 A illustrates the synoptic diagram of two point form Mach-Zender difference interference instrument system, wherein uses a wavelength.This point is stable/and the benchmark interferometer system measures the phase differential by the light wave of two points on the sample 586.Almost reduce the phase noise of interferometer altogether to the geometry of light path.

Collimated laser beam or low-coherence light source are divided into sample 586 paths and reference path by spectroscope 584.Sample beam is by sample 586 and lens L ₁(objective lens) 588 and at the L of last spectroscope 592 fronts ₂(pipe lens) 590.Lens L ₁588 and L ₂590 have focal distance f respectively ₁And f ₂And formation enlargement ratio M=f ₂/ f ₁Microscope.These lens alignings, so that sample 586 and L ₁Distance between 588 is f ₁, L ₁And L ₂Between distance be f ₁+ f ₂, and the imaging plane is positioned at apart from L ₂Distance be f ₂The position.

Reference beam is by being respectively ω with frequency ₁And ω ₂Two acousto-optic modulators 594 driving of radio frequency electromagnetic field, AOM ₁And AOM ₂Iris is used for selecting from AOM ₁+ 1 order diffraction light beam and from AOM ₂-1 order diffraction light beam.So, with frequencies omega ₀At AOM ₁The light wave of incident is with frequencies omega _R=ω ₀+ Ω (Ω=ω wherein ₁-ω ₂) penetrate from second pin hole.This pair of AOM configuration is adopted for the lower heterodyne frequency Ω that obtains about 100kHz.Low heterodyne frequency may be preferred and help optical alignment that for the use of high sensitivity photodetector because change under the very little situation at beam direction, Ω may be set equal to zero.Higher if desired heterodyne frequency can be used single AOM.Treating apparatus such as computing machine 609 and image display 611 and described system communication.

The separated distance of the frequency of the reference beam of frequency displacement equals the lens L of its focal length sum ₃598 and L ₄600 expansions.Signal electromagnet field and benchmark electromagnetism field energy at two imaging planes are described with following formula with plural charactery:

E _S(x，y，t)＝E _S ⁰(x，y)exp[i(φ _s(x，y，t)+φ _N，S(x，y，t)-ωt)] (23)

E _R(x，y，t)＝E _R ⁰(x，y)exp[iφ _N，R(x，y，t)-(ω+Ω)t] (24)

Here x and y are the lateral coordinates along light path, φ _S(x, y t) are the sample phase place of studying, φ _{N, S}(x, y, t) and φ _{N, R}(x, y, t) the interferometer noise in expression sample arm and the reference arm, and E _S ⁰(x, y), E _R ⁰(x y) is the electromagnetic field amplitude profile, and they may be, for example, but be not limited to, Gaussian.

The sample phase _S(x, y t) can use the time dependent refractive index distribution of sample n _S(x, y, z, t) express:

${\mathrm{\&phi;}}_S(x,y,t)={\&Integral;}_{z1}^{z2}{n}_S(x/\stackrel{\&CenterDot;}{M},y/M,z,t)\mathrm{dz}---\left(25\right)$

Wherein z is an axial coordinate, and integration is to finish on the degree of depth of sample.Please note amplification factor M.

Intensity at two imaging planes provides with following formula:

I _±＝|E _S±E _R| ²＝|E _S ⁰| ²+|E _R ⁰| ²

±2|E _S ⁰||E _R ⁰|cos[φ _S(x，y，t)+φ _N，S(x，y，t)-φ _N，R(x，y，t)+Ωt] (26)

Heterodyne signal is with being positioned at position (x ₁, y ₁) and (x ₂, y ₂) two photodiode PD 1604 and PD2 606 survey.Light wave may be collected by optical fiber or pin hole.

The AC component that is detected intensity provides with following formula:

I ₁(t)＝2|E _S ⁰||E _R ⁰|cos[φ _S(x ₁，y ₁，t)+φ _N，S(x ₁，y ₁，t)-φ _N，R(x ₁，y ₁，t)+Ωt] (27)

I ₂(t)＝-2|E _S ⁰||E _R ⁰|cos[φ _S(x ₂，y ₂，t)+φ _N，S(x ₂，y ₂，t)-φ _N，R(x ₂，y ₂，t)+Ωt] (28)

Measure heterodyne signal I with lock-in amplifier or phase detector circuit 608 then ₁With-I ₂Between phase differential.

Ф ₁₂(t)＝[φ _S(x ₁，y ₁，t)+φ _N，S(x ₁，y ₁，t)-φ _N，R(x ₁，y ₁，t)]-[φ _S(x ₂，y ₂，t)+φ _N，S(x ₂，y ₂，t)-φ _N，R(x ₂，y ₂，t)]

＝φ _S(x ₁，y ₁，t)-φ _S(x ₂，y ₂，t)+φ _N，S(x ₁，y ₁，t)-φ _N，S(x ₂，y ₂，t)-φ _N，R(x ₁，y ₁，t)+φ _N，R(x ₂，y ₂，t)

(29)

If suppose that now interferometer noise and lateral attitude are irrelevant, that is,

φ _N，S(x ₁，y ₁，t)＝φ _N，S(x ₂，y ₂，t) (30a)

φ _N，R(x ₁，y ₁，t)＝φ _N，R(x ₂，y ₂，t) (30b)，

What Shi Ce phase differential was the sample phase place at selected point so is poor:

φ ₁₂(t)＝φ _S(x ₁，y ₁，t)-φ _S(x ₂，y ₂，t) (31)

Many photodetectors that can be used in the imaging plane of only obeying physical restriction according to the method for the preferred embodiment of the invention are realized.Photodiode array or photodiode coupled fiber bundle can be used for making the phase place imaging simultaneously of many positions.Any single detector can be selected as " benchmark " detector of measuring the phase differential of all other points with respect to it.

The synoptic diagram of the heterodyne ineterferometer of imaging Mach-Zender is illustrated among Figure 18 B.Device 670 makes the phase imaging that passes through the light wave of sample 673.

${\mathrm{\&phi;}}_S(x,y,t)={\&Integral;}_{z1}^{z2}{n}_S(x/M,y/M,z,t)\mathrm{dz}$

Optical design is similar to the two point form Mach-Zeder heterodyne ineterferometer of describing in conjunction with Figure 18 A, but two changes are arranged: (i) imaging detector (for example, charge-coupled device (CCD) 682) is positioned at one of imaging plane, and (ii) uses photoelectricity light polarization modulator 672 and polaroid 681 to finish the stroboscopic formula and survey.Quantitative phase image obtains with phase-shift interferometry.

Provide with following formula in the time dependent intensity distributions of CCD imaging plane:

I_(x，y，t)＝|E _S±E _R| ²＝

(32a)

|E _S ⁰| ²+|E _R ⁰| ²-2|E _S ⁰||E _R ⁰|cos[φ _S(x，y，t)+φ _N，S(x，y，t)-φ _N，R(x，y，t)+Ωt]

Stroboscopic formula phase interference mensuration is used for making this difference interference figure imaging in the phase sensitivity mode.This needs the detection of " gate " CCD and can finish in several modes.The CCD that strengthens can be by the control booster voltage by gate.Can be at the large aperture of CCD front photovalve as optical gate use fast.In the illustrational system of Figure 18 B, the photoelectricity polarization switch is used for controlling the polarization of the input beam of interferometer.Two kinds of polarizations can be denoted as " in the face " and " face is outer ", corresponding to Figure 18 B.Linear polarizer 681 is placed in CCD imaging device 682 fronts, so that polarized light in the test surface only; The sheet that is polarized of face epipolarized light absorbs or reflects.

The photodiode array of aiming at first imaging plane (if desired, via optical fiber) is used for obtaining the following column signal in the two point form heterodyne ineterferometer:

I ₁(t)＝2|E _S ⁰||E _R ⁰|cos[φ _S(x ₁，y ₁，t)+φ _N，S(x ₁，y ₁，t)-φ _N，R(x ₁，y ₁，t)+Ωt] (32b)

Then, gate-control signal originates from heterodyne signal I ₁, as described below.Electronic comparator is output " high level " when heterodyne signal is positive and positive slope is arranged.This is corresponding to the gate-control signal in phase place 0.In phase shift is pi/2, and the similar signal of π and 3 pi/2s can be by being respectively positive when heterodyne signal and negative slope, negative and negative slope and negative and triggering for generating when positive slope is arranged arranged being arranged.According to the preferred embodiments of the invention, heterodyne signal 687 and gate-control signal 688-691 are illustrated among Figure 18 C.

Then, gate-control signal is used to gate ccd detector continuously.This sequence is subjected to computing machine 685 controls.Have only when gate-control signal is in " high level ", just allow light wave to fall on the CCD.Four exposures corresponding to heterodyne cycle of four gate-control signals rather than equal number are caught by CCD, so that the intensity of correspondence when obtaining four exposures.The stripe pattern of four actual measurements is called as I ₀(x, y), I _Pi/2(x, y), I _π(x, y), I _{3 pi/2s}(x, y).So relative sample phase potential energy calculates with following formula:

${\mathrm{\&phi;}}_S(x,y)=\mathrm{ta}{n}^{-1}\left(\frac{I_{3\mathrm{\&pi;}/2}(x,y)-I_{\mathrm{\&pi;}/2}(x,y)}{I_0(x,y)-I_{\mathrm{\&pi;}}(x,y)}\right)---\left(32c\right)$

Because phase place is being offset between each frame among four frames.Be used for changing phase place and can be used, for example, by quoting as proof it is all instructed the Creath that incorporate at this with other method of calculating phase place, K is at " Phase-Measurement Interferometry Techniques ", Progress in Optics, Vol.XXVI, E.Wolf, Ed., Elsevier SciencePublishers, Amsterdam, 1988, pp, those that describe among the 349-393.In addition, the interferometer noise is as long as it is the noise heterodyne signal I that constant just can have correlationship by benchmark on the plane of delineation ₁(t) be eliminated.Stroboscopic formula phase imaging can be counted as " bucket formula " integrated form, and wherein integration is according to common heterodyne reference signal the time to be finished.

According to the preferred embodiments of the invention, stroboscopic formula phase imaging also can be finished with the twin-beam heterodyne ineterferometer.This needs the low coherence's wavelength that can be surveyed by CCD, for example 850nm.Compare with Figure 19 described below, it also needs the sample beam delivery system is modified as the imaging system shown in Figure 18 D.In this embodiment, the benchmark heterodyne signal that is used for producing four gate-control signals is provided by the optical reference signal.The template of detectable signal may be finished with polaroid by fiber switch or light polarization modulator.

The preferred embodiments of the invention comprise the twin-beam reflection interferometer.The preferred embodiment of twin-beam reflection interference mensuration comprises isolates twin-beam heterodyne LCI.Heterodyne system double beam interferometer 620 is illustrated in Figure 19.Described interferometer is used for measuring from the reflecting light of sample with respect to the phase change that is positioned at the part reflecting surface before the sample.For example, but the phase place of the light wave of the sample reflection of people's energy measurement from the glass flake.As another example, measurement may be with respect to carrying out from the reflection that is placed near the fibre-optical probe top the sample that is studied.

Low coherence such as superluminescent diode (SLD) or multimode laser diode originates and 622 is coupled to by the vacuum feedthrough and enters among the single-mode fiber of vacuum chamber 640.What be closed in this vacuum chamber the inside is the free space heterodyne Michelson interferometer of shock insulation.Low coherence's light beam is launched from optical fiber via collimation lens, is separated by spectroscope 626 then.Two arms of interferometer (being called 1 (656) and 2 (658)) comprise by frequency and are respectively ω ₁And ω ₂The acousto-optic modulator (AOM1 628 and AOM2634) that drives of radio frequency electromagnetic field).In each arm, the first order diffraction beam that just is being offset is selected by pin hole.Light wave is focused on by lens 630 and 636, and mirror M1 632 and two AOM of M2 638 reflected backs then are reflected.Lens are placed in the position of distance A OM and one times of focal length of catoptron.This design allows the AOM retroeflection to proofread and correct the spectrum that continues to keep low coherence's (wide spectrum) light wave.

Because AOM operation in the double light path configuration is with frequencies omega ₀The light wave of incident is being displaced to ω respectively afterwards by arm 1 (656) and 2 (658) ₀+ 2 ω ₁And ω ₀+ 2 ω ₂Two is Ω=2 (ω by the difference on the frequency between the light beam of arm 1 and 2 ₁-ω ₂).

One of catoptron M1 (632) is attached on the translation stage, so that regulate two path length difference Δ l=l between the arm ₁-l ₂After by two arms, beam combination can be counted as the light beam that is separated two pulses by time delay Δ l/c.Be focused by collimator 660 from the reflection of two interferometer arm and return optical fiber and to withdraw from cabin 640.

Light circulator is used for a retroeflection light beam to be separated with incident beam.Light wave is launched by another collimator 662 as free space beam and is focused on the sample 642, therefore the surface of at first reflecting by part 664.Backscattered light wave is collected by same collimator and is surveyed by photodiode 650 after by another light circulator.Regulate the optical delay of Michelson interferometer, so that and match from the reflection of sample S with from the optical path difference Δ s between the reflection of reference surface.When condition Δ L=Δ s maintained within the coherent length L of light source, the heterodyne signal under frequency omega was owing to being detected from the interference between the light wave of surperficial S642 and R 664 reflections.The phase place representative sample reflection of heterodyne signal is with respect to the tolerance of the phase place of benchmark reflection, and described heterodyne signal phase place is measured with respect to the mixing of the electromagnetic field that drives by two AOM and the local oscillator that doubles to provide.Produce heterodyne signal in order to stop by single surface reflection, length Δ s must be in fact greater than coherent length LC.Suppose that thickness of sample is less than thickness of glass Δ s, so that signal relates to glass surface, and do not relate to scattering from sample.

The quantitative description of interferometer is as follows.Consider that at first wave number is k ₀Monochromatic source.Electromagnetic field amplitude at the input end of Michelson interferometer can be described with following formula:

E _i＝A _icos(k ₀z-ω ₀t) (33)

By the electromagnetic field that returns from spectroscope after the AOM is to be used for being provided by the magnetic field sum of two arms of self-interference instrument:

E _m＝E ₁+E ₂＝A _icos(2k ₁l ₁-(ω ₀+2ω ₁)t)+A _icos(2k ₂l ₂-(ω ₀+2ω ₂)t)(34)

K wherein ₁=k ₀+ 2 ω ₁/ c and k ₂=k ₀+ 2 ω ₂/ c.

Dual light beam is the incident beam on the sample now.Make s ₁Be optical range and s to the benchmark reflection ₂It is optical range to the sample reflection.If the reflectivity of benchmark reflection and sample reflection is respectively R ₁And R ₂, and ignore repeatedly reflection, the electromagnetic field from the sample reflection provides with following formula so:

$E_s=A_i\sqrt{R_1}\cos [2k_1\left({\mathrm{<figure-callout\; id="1"\; label="l"\; filenames="A20048002083801151.png,A20048002083801161.png"\; state="\{\{state\}\}">l</figure-callout>}}_{1+}{s}_1\right)-({\mathrm{\&omega;}}_0+2{\mathrm{\&omega;}}_1)t]+A_i\sqrt{R_1}\cos [2k_2({\mathrm{<figure-callout\; id="2"\; label="l"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">l</figure-callout>}}_2+s_1)-({\mathrm{\&omega;}}_0+2{\mathrm{\&omega;}}_2)t]$

$+A_i\sqrt{R_2}\cos [2k_1\left({\mathrm{<figure-callout\; id="1"\; label="l"\; filenames="A20048002083801151.png,A20048002083801161.png"\; state="\{\{state\}\}">l</figure-callout>}}_{1+}{s}_1\right)-({\mathrm{\&omega;}}_0+2{\mathrm{\&omega;}}_1)t]+A_i\sqrt{R_2}\cos [2k_2({\mathrm{<figure-callout\; id="2"\; label="l"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">l</figure-callout>}}_2+s_2)-({\mathrm{\&omega;}}_0+2{\mathrm{\&omega;}}_2)t]---\left(35\right)$

The intensity i that detects _DSquare proportional with the electromagnetic field amplitude:

$i_D\&Proportional;\&lang;{\left|E_s\right|}^2\&rang;=({\mathrm{<figure-callout\; id="1"\; label="R"\; filenames="A20048002083801151.png,A20048002083801161.png"\; state="\{\{state\}\}">R</figure-callout>}}_1+R_2)(1+\cos (2{\mathrm{<figure-callout\; id="0"\; label="k"\; filenames="A20048002083801281.png,A20048002083801282.png"\; state="\{\{state\}\}">k</figure-callout>}}_0\mathrm{\&Delta;l}-\mathrm{\&Omega;t})+$

$2\sqrt{R_1{R}_2}[2\cos \left(2{\mathrm{<figure-callout\; id="0"\; label="k"\; filenames="A20048002083801281.png,A20048002083801282.png"\; state="\{\{state\}\}">k</figure-callout>}}_0\mathrm{\&Delta;s}\right)+\cos \left(2k_0(\mathrm{\&Delta;l}+\mathrm{\&Delta;s}-\mathrm{\&Omega;t})\right)+\cos \left(2k_0(\mathrm{\&Delta;l}-\mathrm{\&Delta;s}-\mathrm{\&Omega;t})\right)]$ (36)

Wherein optical frequency vibration is left in the basket, and wave-number migration Ω/c of causing of supposition frequency shift (FS) and the anti-phase of path length difference Δ s and Δ l relatively are to ignore.

In order to set up low coherence (broadband) light source model, suppose that it has Gauss's power spectrum density, wherein the cardiac wave number is k ₀And long half maximal value (FWHM) spectral bandwidth of all-wave is Δ k.

$s\left(k\right)=\frac{2\sqrt{\mathrm{<figure-callout\; id="2"\; label="ln"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">ln</figure-callout>}2}}{\mathrm{\&Delta;k}\sqrt{\mathrm{\&pi;}}}\exp [-{\left(\frac{k-{\mathrm{<figure-callout\; id="0"\; label="k"\; filenames="A20048002083801281.png,A20048002083801282.png"\; state="\{\{state\}\}">k</figure-callout>}}_0}{\mathrm{\&Delta;k}/\left(2\sqrt{\ln 2}\right)}\right)}^2]---\left(37\right)$

To low coherence's radiation detection to intensity by found that in spectrum distribution upper integral monochrome:

$\widetilde{i_D}={\&Integral;i}_D\left(k\right)S\left(k\right)\mathrm{dk}=({\mathrm{<figure-callout\; id="1"\; label="R"\; filenames="A20048002083801151.png,A20048002083801161.png"\; state="\{\{state\}\}">R</figure-callout>}}_1+R_2)(1+F\left(\mathrm{\&Delta;l}\right)\cos (2{\mathrm{<figure-callout\; id="0"\; label="k"\; filenames="A20048002083801281.png,A20048002083801282.png"\; state="\{\{state\}\}">k</figure-callout>}}_0\mathrm{\&Delta;l}-\mathrm{\&Omega;t})$

$+2\sqrt{R_1{R}_2}[2F\left(\mathrm{\&Delta;s}\right)\cos \left(2{\mathrm{<figure-callout\; id="0"\; label="k"\; filenames="A20048002083801281.png,A20048002083801282.png"\; state="\{\{state\}\}">k</figure-callout>}}_0\mathrm{\&Delta;s}\right)+F(\mathrm{\&Delta;l}+\mathrm{\&Delta;s})\cos \left(2k_0(\mathrm{\&Delta;l}+\mathrm{\&Delta;s}-\mathrm{\&Omega;t})\right)$

$+F(\mathrm{\&Delta;l}-\mathrm{\&Delta;s})\cos \left(2k_0(\mathrm{\&Delta;l}-\mathrm{\&Delta;s}-\mathrm{\&Omega;t})\right)]$ (38)

Wherein

$F\left(x\right)=\exp [-{\left(\frac{x}{l_c/\left(2\ln 2\right)}\right)}^2]---\left(39\right)$

It is the light source coherence function of selected spectral density.L here _cIt is coherent length

$l_c=\frac{4\left(\ln 2\right)}{\mathrm{\&Delta;k}}=\frac{2\left(\ln 2\right){\mathrm{\&lambda;}}_0^2}{\mathrm{\&pi;\&Delta;\&lambda;}}---\left(40\right)$

If path length difference is selected like this, consequently Δ l=Δ s within coherent length, and Δ l＞＞l _c, fixed signal has following form according to the domination time so:

$\widetilde{i_D}\left(\mathrm{AC}\right)=2\sqrt{R_1{R}_2}\left[F(\mathrm{\&Delta;l}-\mathrm{\&Delta;s})\cos \left(2k_0\right(\mathrm{\&Delta;l}-\mathrm{\&Delta;s}-\mathrm{\&Omega;t})\right)]---\left(41\right)$

By measuring the phase place of this signal with respect to local oscillator 652LO=cos (Ω t), the change of Δ s can be measured.Note that the change in order to stop phase noise to pass through Δ l influences measurement result, the isolation of Michelson interferometer is absolutely necessary.

Figure 20 illustrates the low coherence's interferometer of dual benchmark heterodyne of isolation according to the preferred embodiments of the invention.Interferometer is used for measuring from the light wave of selected sample degree of depth reflection with respect to the phase place from the scattering of the different sample degree of depth.Because do not need the glass-reflected surface, this assembly is better than fairly simple double beam interferometer.This system is desirable for somatometry.Dual benchmark Michelson interferometer can be used for making nervous activity imaging on three-D volumes in enough thin or transparent sample.Described system can be used for studying the growth of neural network.

Be separated among top and the following path by fiber coupler 706 from the light wave of low-coherence light source 702.Top class of paths is similar to the double beam interferometer of above describing in conjunction with Figure 19, and frequency displacement is ω ₁Double light path AOM replace being positioned at now sample among the following path of described interferometer.Two electromagnetic fields are reconfigured in another fiber coupler 742.Photodiode the 746, the 748th is arranged by two balanced modes.

Under the monochromatic source situation, the quantitative description of interferometer is as follows.The electromagnetic field in top path can be write:

E ₁＝A _icos(2k ₀l ₁-(ω ₀+2ω ₁-2ω ₃)t)+A _icos(2k ₀l ₂-(ω ₀+2ω2-2ω ₃)t)

(42)

And following path is (to suppose that once more sample is included in position s ₁And s ₂Two reflections):

${\mathrm{<figure-callout\; id="2"\; label="E"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">E</figure-callout>}}_2=A_i\sqrt{R_1}\cos \left[{2k}_0{s}_1-{\mathrm{\&omega;}}_{0}t\right]+A_i\sqrt{R_2}\cos \left[2k_0{s}_2-{\mathrm{\&omega;}}_{0}t\right]---\left(43\right)$

The path of having supposed fiber optic cables equates between two arms.With frequency be ω ₃The catoptron 740 that is associated of AOM 736 can be by translation, so that path equates.

The AC component of photo detector signal provides with following formula:

$i_D\&Proportional;\&lang;{|{\mathrm{<figure-callout\; id="1"\; label="E"\; filenames="A20048002083801151.png,A20048002083801161.png"\; state="\{\{state\}\}">E</figure-callout>}}_1+{\mathrm{<figure-callout\; id="2"\; label="E"\; filenames="A20048002083801151.png,A20048002083801171.png"\; state="\{\{state\}\}">E</figure-callout>}}_2|}^2{\&rang;}_{\mathrm{AC}}=\sqrt{R_1}[\cos (2k_0(l_1-s_1)-{\mathrm{\&Omega;}}_{13}t)+\cos (2k_0(l_2-s_1)-{\mathrm{\&Omega;}}_{23}t)]$

$+\sqrt{R_2}[\cos (2k_0(l_1-s_2)-{\mathrm{\&Omega;}}_{13}t)+\cos (2k_0(l_2-s_2)-{\mathrm{\&Omega;}}_{23}t)]$ (44)

Ω wherein ₁₃=2 (ω ₁-ω ₃), and Ω ₂₃=2 (ω ₂-ω ₃).With regard to Gaussian spectrum distributed, the polychrome situation provided:

${\tilde{i}}_D={\&Integral;i}_D\left(k\right)S\left(k\right)\mathrm{dk}\&Proportional;\sqrt{R_1}[F(l_1-s_1)\cos (2k_0(l_1-s_1)-{\mathrm{\&Omega;}}_{13}t)$

$+F(l_2-s_1)\cos (2k_0(l_2-s_1)-{\mathrm{\&Omega;}}_{23}t)]$

$+\sqrt{R_2}[F(l_1-s_2)\cos (2k_0(l_1-s_2)-{\mathrm{\&Omega;}}_{13}t)+F(l_2-s_2)\cos (2k_0(l_2-s_2)-{\mathrm{\&Omega;}}_{23}t)]---\left(45\right)$

If within coherent length, l ₁≈ s ₁, l ₂≈ s ₂, and Δ l, Δ s＜＜l _c, major event is so

${\tilde{i}}_D\&Proportional;\sqrt{R_1}F(l_1-s_1)\cos (2k_0(l_1-s_1)-{\mathrm{\&Omega;}}_{13}t)+F(l_2-s_2)\cos ({2k}_0(l_2-s_2)-{\mathrm{\&Omega;}}_{23}t)]---\left(46\right)$

Next, these two frequency components are combined in frequency mixer, and pass filter is selected difference frequency Ω ₁₂=Ω ₁₃-Ω ₂₃=ω ₁-ω ₂:

$X=\sqrt{R_1{R}_2}F(l_1-s_1)F(l_2-s_2)\cos ({2k}_0(l_1-l_2-(s_1-s_{2)})-{\mathrm{\&Omega;}}_{12}t)---\left(47\right)$

Then, phase-sensitive detection device measuring-signal is with respect to driving the mixing of electromagnetic field and the Ω that doubles to produce by AOM ₁₂Under the phase place of local oscillator.The phase place of this actual measurement is φ=2k ₀(Δ l-Δ s).

Phase shifter is used for offsetting the derivative characteristic that may ignore it has some influences to phase measurement interferometer noise.The phase place of photodiode signal component

$\sqrt{R_1}F(l_1-s_1)\cos ({2k}_0(l_1-s_1)-{\mathrm{\&Omega;}}_{13}t)---\left(48\right)$

Measured and be used as error signal, will be from s ₁Reflection lock onto invariable phase place by phase shifter.

In the embodiment of using authentic sample, except scatter distributions, will there be two reflections.By setting the reference arm position, interferometer measurement is from the phase differential between the light wave of two different depth scatterings.

Measure the phase place of optics heterodyne signal in conjunction with the embodiment that Figure 19 describes with respect to the electric signal that produces by the mixing of acousto-optic modulator (AOM) radio frequency electromagnetic field.The numerous noise source that is associated with embodiment is arranged.They comprise perhaps owing to from the AOM heating of radio frequency electromagnetic field be about several minutes an about wavelength (1 λ) slow drift and at the phase noise of 60Hz and 120Hz.In addition, amplitude appears in the AOM tuning voltage and perhaps owing to line noise.Other source of wide band amplitude that optical fiber in the outside, cabin changes when moving and phase noise is most possible owing to the polarization mode dispersion in the light circulator (PMD).

The preferred embodiments of the invention reduce to greatest extent and preferably eliminate noise and comprise that optical reference is measured or use the AOM tuning voltage that the precise voltage power supply provides or use the fiber optics parts of keeping polarization to reduce drift and noise as an alternative.Figure 21 illustrates the preferred embodiment of the optical reference interferometer that reduces noise to greatest extent.Described embodiment solves drift that system experienced and the noise problem of describing in conjunction with Figure 19.With reference to the illustrational embodiment of Figure 21 is the heterodyne system double beam interferometer.Interferometer is used for using may be as the reference measurement of crosswise spots different on sample object or the individual element phase change from the reflecting light of sample.Low coherence light source 762 such as SLD is coupled to by the vacuum feedthrough and enters among the single-mode fiber of vacuum chamber 782.Heterodyne Michelson interferometer is described as Figure 19 and is operated.Return two optical fiber and withdraw from this cabin from collimated instrument 766,792 focusing of the reflection of two interferometer arm.Additional optical distance provides the benchmark as different crosswise spots on individual element.The back-scattered light wavelength-division is collected by two collimators 788,790 and is surveyed by two photodiodes 796,800 after passing through light circulator 794 and 798.

In dual benchmark interferometry embodiment, lateral fiducial point and the measurement of reflection-reference phase are combined.It is desirable to, reference point and sample object all are positioned on the same glass.Any inclination, vibration and/or the bulking effect of eliminating in the cover glass there is additional benefit.As shown in figure 22, the phase place of actual measurement is:

φ(t)＝(φ ₁-φ ₁′)-(φ ₂-φ ₂′) (49)

The preferred embodiment of dual benchmark interferometer includes the photodetector of similar gain and frequency response, so that eliminate noise.In addition, keep the component of polarization and optical fiber and can be used for handling polarization effect in the optical fiber.Specifically, the polarization mode dispersion in the light circulator produces between two cross polarization and causes and can keep the amplitude that the parts of polarization alleviate and the variable delay of phase noise by use.In preferred embodiments, digital pass filter is used for handling the harmonic wave of finding in optical signalling.

Illustrate to Figure 23 A graphic formula the voltage of the piezoelectric transducer (PZT) that is used for demarcating the system of describing in conjunction with Figure 21.PZT demarcates assembly in Figure 25 A illustrated.

With the corresponding phase change of the displacement of catoptron 888 be that graphic formula ground is illustrational in Figure 23 B.This phase change is corresponding to the demarcation variable in distance of 27nm.

Figure 24 graphic formula ground is that unit illustrates the noiseproof feature that is associated with the illustrational interferometer of Figure 21 in cycle T.T. of 50ms with the radian, and wherein two of interferometer arms equate.

Figure 25 A and 25B are the signal expression that is used for the demarcation assembly of sample signal and reference signal according to the preferred embodiment of the invention.Changing along with PZT at interval between catoptron and spectroscope.The motion of PZT is that (He-Ne or Ti: emission sapphire) is demarcated by the monitoring light source.

The preferred embodiments of the invention relate to the low coherence's interferometer of twin-beam that system comprises the non-cpntact measurement of the slight movement that is used for finishing the weak reflecting surface such as neural shift movement during action potential.Nerve fibre presents outwards side displacement rapidly during action potential.This " expansion " phenomenon that usually flows into aixs cylinder owing to water is observed in the crab nerve at first, be afterwards many other invertabrate and vertebrate experimental specimen in observed.All nerves expand and observe optical sensor or the piezoelectric sensor of still relying on so far with neural actual contact.Being used for measuring the neural non-contact optical method that is shifted can eliminate the artifact relevant with contact and allow the native state simultaneously imaging of the activity of many nerves with them.

The preferred embodiments of the invention comprise low coherence's interferometer of twin-beam heterodyne system and the application in the bulking effect of measuring the lobster nerve tract thereof.Existing with the success as yet of the neural method that expands of interferometer observation, because sensitivity is hanged down and can't be surveyed the motion that action potential any and frog or lobster nerve is associated.Recently, having used the transmitted light wave interferometer successfully to measure neural refractive index during action potential changes.

Measurement is about several nanometers in the millisecond time range neural displacement want can from the antiradar reflectivity surface recording fast with stable interferometer measuring system.According to embodiment preferred, the double-beam system of being made up of single-mode fiber and free space element is illustrated among Figure 26.From with the light wave collimation of the optical fiber of superluminescent diode 922 (Optospeed SLD, the centre wavelength of FWHM is 1550nm, bandwidth is 40nm) coupling after enter be included in alignment in the double light path configuration and be ω=110.1MHz and ω by frequency ₂The Michelson interferometer of the acousto-optic modulator (AOM) 946,952 that the radio frequency electromagnetic field of=110MHz drives.The catoptron that is installed on the translation stage allows to be controlled at round optical path difference Δ L between two interferometer arm.Light wave is by optical fiber collimator turnover Michelson interferometer.

From the output of each mouthful among two mouths of Michelson interferometer by two dual light beams that low coherence's electromagnetic field is formed by different frequency displacements and variable delay.One of dual light beam is the incident beam on the neural cabin assembly (describing in detail in Figure 27), and another is the incident beam on the benchmark event.Each all comprises two reflecting surfaces that separated by adjustable distance neural assembly and benchmark event, and by alignment so that with its optical fiber separately of incident light wave reflected back.One of these surfaces almost are at air and do not have interface between the coated glass.Second reflection comes the interface between comfortable air and the neural surface in sample.

Δ L _SWith Δ L _RBe respectively from the round optical path difference between the reflection of the surface 1 of sample and benchmark event and 2.Regulate various component so that three path Δ L, Δ L _SWith Δ L _RAll equal within the coherence length of laser.When condition satisfies, photodetector 932,962 (new focusing 2011) record since (1) cross the arm 1 of Michelson interferometer and the light wave that reflects from the light wave of surface 2 reflections of sample (or benchmark event) and arm 2 that (2) cross the Michelson interferometer and from the surface 1 of sample (or benchmark event) between the frequency that causes of interference be Ω=2 (ω ₁-ω ₂The heterodyne signal of)=200kHz.Phase differential between two heterodyne signals (the nearly multiple of 2 π) is φ (t)=k ₀[(Δ L _S-Δ L)-(Δ L _R-Δ L)]=k ₀(Δ L _S-Δ L _R), k wherein ₀It is the center wave number in source.Be subject to most phase noise influence amount---Michelson Δ in path delay L is deleted in this variate method.Be used for making the power that detects to reach maximal value with the light circulator 926,930,960 of polarization irrelevant and stop reflecting light to enter the Michelson interferometer once more.Polarization Controller (show) is used for reducing to greatest extent the effect of the polarization mode dispersion in the fiber optics component.

For measure phase difference φ (t), the output of photodetector is by the speed digitizing with 5,000,000 samples of per second of 12 A/D card (PCI-6110 of American National instrument company).Instruction sequence in the computing machine is expressed as facing surfaces displacement d (t)=φ (t)/2k by two phase difference between signals of Hilbert transformation calculations and this phase deviation ₀

In order to check interferometer to finish displacement measurement, neural assembly is used piezoelectric transducer and replaces by sinusoidal curve modulating resonance chamber plane Fabry-Perot resonator cavity at interval with 300Hz frequency and 27nm amplitude.When resonator cavity was scanned in some micrometer ranges, the amplitude of double beam interferometer was consistent well with the numerical value of determining by the transmission of monitoring 632.8nm he-Ne laser light beam with frequency measurement.

According to embodiment preferred, dissected and be placed on the neural cabin that forms with acryl resin machining shown in Figure 27 from the step nerve (～1mm diameter ,～50 millimeters long) of U.S. lobster (America lobster).This cabin comprises five containers that are full of the lobster brine solution, and the nerve between each container is surrounded by the vaseline insulation course, so that resistance maximum between the container.Stimulation isolator is sent the current impulse (0-10mA, duration 1ms) of variable amplitude so that excite nerve by stimulating electrode.The electric current that stimulation isolator is sent is owing to lead perhaps than much bigger by neural real current by the parallel electricity of salt solution.The complex action potential that produces in nerve is surveyed with a pair of recording electrode 998a, 998b, and with 104 amplifications that gain.In center pit, nerve is placed on the little glass platform, so that it is not immersed in the brine solution.During dissection and data aggregation, neural moist with the salt solution maintenance.

Figure 28 A and 28B illustrate according to the current potential of the preferred embodiments of the invention in a test and neural optical method measured displacements.The spike at time zero place is that artificial the stimulation caused in electric signal.The peak of the action potential of a plurality of aixs cylinders in a series of description nerve tracts is followed in its back.Optical signalling shows and highly to be approximately the peak that 5nm, FWHM duration are approximately 10ms that its direction is corresponding to the upper surface displacement.Optical signalling is showed single peak, rather than as electric signal a plurality of spikes; This may be because single-phase (monadic symbols) characteristic of displacement signal causes.The rms noise of displacement measurement is approximately 0.25nm for the 1kHz bandwidth.

Displacement is observed and change to 8nm for the 5mA stimulation amplitude from 0nm in only about half of neural sample.Big changeableness can reflect the difference aspect nerve itself or preparation routine.Similar displacement amplitude has also had the report of the nerve that uses crab and cray.About～10 less displacements are observed in the neural expansion current research of the lobster of using optical lever, and this may reflect the artefact of technology.

In order to control the artefact such as the nerve heating that causes owing to stimulating current from the thermal expansion of Ohmic heating, the peak electricity signal of single nerve and displacement signal are measured (as shown in figure 29) when changing stimulating current.Electric signal and displacement signal present almost same threshold current (approximately 1.5mA) and saturation current (approximately 5mA), thereby hint that observed displacement is associated with action potential.On the contrary, ohm effect will be characteristic and with saturated irrelevant with the secondary dependence with electric current.Therefore, provide the neural displacement research that to control artificial stimulation according to the preferred embodiments of the invention.Embodiment preferred comprises finishes the low coherence's interferometer of heterodyne contactless first and the neural dilatometry of interferometry first.According to the preferred embodiments of the invention, neural just other aixs cylinder imaging and analysis of biophysics mechanism of expanding.The low coherence's interferometer of twin-beam may have many other application aspect the nano level motion of measuring living cells.Other embodiment can comprise with the interferometer being the microscope that the single neuronic mechanical alteration that is associated with action potential is surveyed on the basis.Relevant interferometry also is used for measuring the change of cell volume in the quilt cell monolayer of cultivating.

Figure 30 illustrates the optical design of the scanning system of double beam interferometer according to the preferred embodiments of the invention.The catoptron that is installed on the motorization galvanometer 1024,1030 is placed on the Fourier plane of imaging system and allows light beam with the inswept sample of constant angle.Catoptron is with about 30Hz, 1-2 degree amplitude (50-100nm on sample) scanning.Galvanometer can be set up grating or scanning by Lissajous Figure 105 2.

Figure 31 illustrates according to the preferred embodiments of the invention galvanometer position and uses the phase data of Lissajous scanning from blank cover glass collection.Total visual field approximately is 100 microns.Z-axis is the phase place of actual measurement.The noise profile is about 25mrad on 1kHz.

Figure 32 A and 32B illustrate the color map according to the phase image of the preferred embodiments of the invention retroeflection and intensity (amplitude) image.The beam flying data are collected from blank cover glass.The noise profile is about 25mrad on 1kHz.Power is because misalignment that takes place when light beam leaves optical axis and moves and pruning are the highest at the center.Stain in the image is corresponding to scheming not accessed pixel with Lissajous.

Figure 33 schematically illustrates the focus issues that solves by embodiment of the present invention.Cell 2002 is placed on the microscopical glass cover slide 2004 of twin-beam.The solid line representative focuses on the light beam on surface on glass and the cell.The dotted line representative is from the light beam of the bottom reflection of glass.Phase place-benchmark interferometer measuration system is the double beam interference measuring method according to the preferred embodiment of the invention, and need not only collect from the light wave of interest sample scattering but also collect from the reflection of the fixing reference surface that is positioned at the sample front.The scattering that benchmark and sample are collected in the reply of the sample axial location different with benchmark effectively simultaneously is especially for the optical system of high-NA.Two kinds of solutions of focal issue are to be provided by two kinds of systems that allow among the preferred embodiment to collect the light wave of sample and benchmark under for 0.50 situation effectively in numerical aperture.At first, the bifocal lens system brings sample surfaces and reference surface into focus simultaneously by numerical aperture being divided into by the marginal ray and the paraxial rays of the different part of bifocus optical element mean curvature.The bifocal element can constitute by polish the central area on the convex surface of plano-convex lens.Bifocal lens is placed near the beam flying that allows the microscopical Fourier plane via the mirror galvanometer that is used for the sample imaging.

Figure 34 illustrates the bifocal lens design according to the preferred embodiment of the invention.Bifocal lens is placed on the object lens front rather than the back is to be relatively easy to.Described embodiment makes translation become easy.

Figure 35 illustrates the another kind design according to the bifocal lens system of the preferred embodiments of the invention.Bifocal lens system 2050 by bifocal lens being placed on imaging system Fourier plane or this plane near allow beam flying.Light beam 2068 is paraxial beams, and light beam 2070 is edge light beams.Described optical property is constant to the single order diagonal beam.

Figure 36 illustrates the calculating of the optimum distance between lens f3 (bifocal) and the f2, so that spacing between two focal lengths of object lens back equals Δ=100 micron according to the preferred embodiments of the invention.Described optical design provides by ray trace.

Figure 37 illustrates the manufacturing according to the bifocal lens of the preferred embodiment of the invention.Bifocal lens can be made by the core of polishing plano-convex lens.Remove very little thickness of glass (approximately 2-10 μ m).Little change causes the path length difference between signal and the benchmark.

Figure 38 illustrates the actual measurement retroeflection intensity of passing through light circulator when object lens according to the preferred embodiments of the invention when the glass cover slide scans.Be separated about 100 microns from the back side and positive reflection.Do not have overlapping and therefore not interference.

Figure 39 just has according to the preferred embodiments of the invention from the relation that illustrates with regard to the bifocal lens of the individual reflection of catoptron between retroeflection intensity and the object focal point position.Use bifocal lens f3, (when object lens position is scanned) is divided into two from the unimodal of catoptron, corresponding to paraxial beam and the edge light beam of different object focal point positions at focusing mirror.Peak-to-peak spacing depends on the distance between lens f2 and the f3.In the present embodiment, spacing is about 100 microns.

Retroeflection intensity when Figure 40 illustrates and uses bifocal lens according to the preferred embodiments of the invention under the situation that the reflection of two-sided cover glass is arranged and the relation between the object focal point position.This figure puts two previous figure together, and four main peaks and several less peak are provided.

Figure 41 illustrate according to the preferred embodiments of the invention when the distance between f2 and the f3 be adjusted to and the front and back glass surface between spacing relation between retroeflection intensity and object focal point position when being complementary.Marginal ray focuses on the back side, back, focuses on the preceding back side at the same position paraxial rays.This provides the big peak that can see at the center.

Figure 42 illustrates the extra less peak that produces owing to the coupling according to the preferred embodiments of the invention paraxial beam and edge light beam.These extra peak energy peak-to-peakly obtain explaining to the edge light beam coupling from paraxial by accurately occurring in two halfway.The amplitude at extra peak depends on optical alignment to heavens and can be reduced to minimum and preferably be eliminated by the fine setting optical system.

Figure 43 A illustrates according to the twin-beam probe that reference surface arranged of the preferred embodiments of the invention as integrated component.This probe is made up of optical fiber collimator 2382 and gradually changed refractive index (GRIN) lens 2390.The uncoated rear surface of grin lens provides the benchmark reflection.Owing to do not need reference surface separately, so this probe is very suitable in vivo using.In order to finish two-dimensional phase imaging or three-dimensional confocal phase imaging, this probe may be installed on the quick scanning piezoelectric transducer.Described embodiment provides the fibre-optical probe of aiming in advance that is used for displacement measurement easily.This probe has high-NA (NA), in the scope of about 0.4-0.5, provides from the effective light wave of scattering surface and assembles.Whole reference surface solves the problem that the depth of field causes.The probe of described preferred embodiment replaces the set of complex optical components and is suitable in vivo using.

Figure 43 B is illustrational to be another preferred embodiment of optical-fiber type two-beam interference instrument probe 2381.In this example, the benchmark reflection is provided by optical fiber interlayer end 2385.In this case, end 2385 is polished with fiber axis and meets at right angles.Optical fiber 2383 is installed in the glass collar 2389 that injects shell 2387.The light wave that optical fiber connector 2385 sends is with gradually changed refractive index (GRIN) lens focus, and these lens have about 0.29 or 0.3 pitch in this example.Be approximately equal to 3.5 enlargement factor M by having of alignment optical fiber, so that the numerical aperture of optical fiber (NA=0.13) and grin lens (NA=0.50) is complementary.Light beam focuses on the sample, and sample is apart from about 300 microns of the distal surface of popping one's head in this example.

Figure 43 C is the bright-field microscope image from two nerve fibres (aixs cylinder or dendritic crystal) of mouse hippocampus culture.Figure 43 D is the expression as the heterodyne signal amplitude of the function of position that uses the actual measurement of bifocal twin-beam microscope scanning samples.

Figure 43 E is a reflected phase will image of seeing same sample in Figure 43 D.

Figure 44 be according to the preferred embodiments of the invention be used for studying during the action potential in nerve the synoptic diagram 2400 of the geometric twin-beam probe of observed displacement effect.By changing the angle of probe, the displacement of energy measurement different directions.

Figure 45 illustrates the twin-beam probe system that can be used for imaging according to the preferred embodiments of the invention by scanning head or sample.For fear of introducing the artefact that causes owing to the sample motion, scanning head is preferred.Probe is because its (approximately 2-3 gram) in light weight can be by (approximately 1kHz) scanning very fast.Because reference surface is integrated in the probe, (intensity and phase place) three-dimensional confocal imaging is possible.Dual light beam probe system energy measurement transmission or the reflection enumerated.Embodiment preferred comprises sweep type twin-beam probe microscope, so object lens are high stability to the distance of sample.

Figure 46 A-46C illustrates respectively according to the preferred embodiments of the invention and uses the intensity image that the human cheek epithelial cell (or two superpose cells) of the drying of bifocal twin-beam microscope from the glass cover slide that is placed on anti-reflection coating obtains, the actual measurement phase image and the bright field image of backscattering light wave.These images are to have the microscope stage of electric translation device to produce by scanning.The image of Figure 46 A and 46B (visual field that 130 microns * vertical direction of horizontal direction is 110 microns, scanning are 100 * 100 pixels) shows the amplitude and the phase place of heterodyne signal respectively.Figure 46 C is the bright field image (visual field is approximately 60 microns * 40 microns) of same cheek cell.This phase image show be less than a ripple contrast this may reflect owing to contact the pattern of almost smooth cell lower surface with glass basis.

Figure 46 D-46G illustrates the microscopical contour curve of the illustrational twin-beam of Figure 43 and measures ability, and the concave surface of the plano-convex lens of 25 millimeters focal lengths is placed on the cover glass shown in Figure 46 E, and the coating of anti-1.5 micron wave lengths reflection is arranged at the top of described cover glass.Figure 46 D illustrates the intensity image of lens center part.Figure 46 F is the phase mapping figure of reflecting light.Figure 46 G illustrates the xsect of phase image, and phase place is launched by quadratic fit.The coefficient of second order term is corresponding to 11.7 millimeter the radius-of-curvature consistent with known lens surface curvature.Outlying point in intensity image and phase image may be that grit or the depression on the lens causes.

Figure 47 A-47E illustrates the phase-shifting interference measuring ratio juris, comprises interferogram and collects the synoptic diagram of the distinct methods of picture such as phase place stepping (Figure 47 B and 47C) and barrel formula integration (Figure 47 D and 47E) according to the preferred embodiments of the invention.This method comprises the minimum value and the calculating optical phase shift of phase modulation, three pictures of record.

Figure 48 A-48C illustrates the principle according to the stroboscopic formula difference interference measuring system of the preferred embodiment of the invention.In order to provide low-down phase noise, this system comprises the acoustooptic modulation that causes being similar to the continuous phase oblique ascension of barrel formula integration except the switching of bucket formula relates to relevant noise floor heterodyne signal.Otherwise stroboscopic formula heterodyne ineterferometer provides continuous measurement, because the pause that does not have the displacement of mechanical type catoptron to cause.

Figure 49 illustrates the synoptic diagram according to the stroboscopic formula twin-beam heterodyne ineterferometer 2570 of the preferred embodiments of the invention.From the collimated photoelectricity light polarization modulator 2594 that enters then of the light wave of double beam interferometer.Light wave reflects from spectroscope 2582, by being two lens 2580,2576 (f1 and f0, object lens) of telescope configuration, then as collimated light beam irradiation sample.Retroeflection light wave from sample (for example, cell 2573) and cover glass rear surface is collected on the CCD 2586 by object lens and f1.Lens f1 is adjusted to apart from focal length of CCD with apart from 2578 focal lengths of Fourier plane (FP).Object lens f02576 is apart from focal length of Fourier plane with apart from focal length of sample equally.The photoelectricity light polarization modulator serves as optical switch fast with polaroid 2584 combinations in the CCD front.The photoelectricity light polarization modulator is to implement gate according to conduct from the phase place of the reference signal of the heterodyne signal of the benchmark event in the double beam interferometer.The SLD of 820nm is used to preferred embodiment.Light wave on the sample 2573 is collimated, therefore gets rid of any focal issue in advance.

Figure 49 B and 49C illustrate according to the preferred embodiments of the invention and show that twin-beam probe shown in Figure 43 focuses on the data of the phase noise that causes on the static glass surface.

Show and the system described challenges to attempting in typical example light wave focused on axially to be separated on about 100 microns reflecting surface and the sample in conjunction with Figure 26.Under the situation of numerical aperture＞0.1, this is more much bigger than the depth of field.It is possible changing that bifocal optical system or probe designs address this problem, yet, the bifocal system worse low collection efficiency that becomes when the high-NA objective of employing is arranged usually, and can produce edge effect from the diffraction of shaft portion from lens.Probe designs is subjected to surperficial benchmark and sample to separate the restriction that is limited in 0.5 this fact with NA when adopting grin lens.

Figure 50 A is illustrational be light wave from two paths before being introduced on the sample earlier along altogether to the system 2700 of light path merging.Light wave from path 1 focuses on the surface 1, and focuses on the surface 2 from the light wave in path 2.

The interferometer that this feature is incorporated into is illustrated in the double-beam system 2800 of Figure 50 B.The polarization spectroscope 2811 that system 2800 has 2801, two mobile mirrors of light source 2803,2805 and handle to guide sample 2807 surfaces and benchmark 2809 surfaces into from the light wave of two paths.The direction of polarization component clearly is illustrated among Figure 50 C.Each arm of interferometer is introduced on the surface.Therefore light wave is more effectively used.Light beam is separated to focus on, and does not have the edge effect from bifocal lens.Do not have the compromise of consequent numerical aperture aspect, and this system's transaction can be configured in and not need the optical fiber coupling in the free space, except optical fiber coupling optionally from light source.This can improve the light wave collection efficiency.

The quantitative phasecontrast microscope of usage space light wave modulation:

On the other hand, the invention provides phasecontrast microscope with and the phase-shift interferometry microscopic system and the method that combine.System and method of the present invention can be applied to transmission geometry and reflection geometry.In various embodiment, described method and system is being used for phase place between the ripple of different space frequency and the ripple that the different space frequency of same point on the sample is risen in skew to light path altogether.

The phase place of optical field has been used to provide the wavelet that needs in many application long accuracy for many years.For example, use the principle of phasecontrast microscope to become observable as all mistakes of department of biology of weak in essence scatterer.Interferometry is a kind of approach that obtains phase information, so, be that purpose has been developed various interfere measurement technique to regain the phase place that is associated with sample in the several years in the past.Though the technology such as phase contrast and Nomarski microscope is very useful and be popular method, only use optical phase as setting off by contrast instrument, do not provide about other big or small quantitative information.

On the other hand, phase shift technology can be determined phase information quantitatively, and is suggested in the various interferometry scheme many decades in the past.Dock based on the differential phase-contrast technique of polarization optics with common optical coherence tomography.Bucket formula integrated technology as the particular case of phase-shifting interference measuring method, also has been used to the two-dimensional phase imaging.Yet most of such interferometers need form two physically separated light beams, and this makes them be subject to uncorrelated neighbourhood noise influence.In order to eliminate noise on one's own initiative, this problem often needs specific measure.Phaselocked loop has been used to this purpose.Needed is microscopic system and the method that reduces or eliminates from the uncorrelated noise of interferometry signal.

System and method of the present invention uses altogether and makes the light wave of the different space frequency of rising in sample relevant to light path.In various embodiment, the system and method that the present invention proposes provides does not have the sample of uncorrelated environment phase noise phase image in fact.In addition, in preferred embodiments, even method of the present invention exists the phase place singular point also can obtain phase image when using low coherence's lighting source.

In various preferred embodiment, the invention provides environment phase place insensitive for noise and the instrument of very accurate and stable phase information can be provided in time shutter scope arbitrarily.In various embodiment, the present invention is based on the iamge description as interferogram.An example of this description is an Abbe's theory of image formation.Each point in the plane of delineation all is counted as with respect to overlapping (interference) of optical axis with the ripple of different angles propagation.If we are considering as the benchmark of interferometer from the zeroth order scattering of sample, this image can be counted as the zeroth order electromagnetic field and leave interference between the electromagnetic field that optical axis propagates so.

Figure 51 A-5D is that the signal of the various different characteristics of such iamge description is expressed.Figure 51 A is that the signal of the interferogram 1102 that formed by high spatial frequency component 1104 and zeroth order component 1106 expresses 1100.Figure 51 B is that the signal of the interferogram 1112 that formed by low frequency component 1114 and zeroth order component 1106 expresses 1110.Figure 51 C expresses 1120 by the light beam 1124 of spatial frequency broadness in the signal of the diffraction spot that overlaps to form 1122 of the plane of delineation 1126.Figure 51 D expresses 1130 by narrower spatial frequency spectrum 1134 in the signal of the diffraction spot 1132 of the broad of the plane of delineation 1126 generations.In addition, for example, the component of zeroth order component and higher-order can be counted as DC component and AC component.

The amplitude of field and the intensity in the plane of delineation can be expressed as in imaging plane:

I _image∝cos( ₁- ₀) (51)

E wherein _ImageRepresent the electric field amplitude of light wave certain point on imaging plane, represents the phase place of light wave, I _ImageRepresent the intensity of light wave certain point on imaging plane, and subscript 0 and 1 is wherein represented zeroth order component and the high-order component that is used for equation 50-56 respectively, for example, single amplitude is considered.Equation 51 illustrates for the phase change Δ = that compares very little with π on sample ₁- ₀, the intensity in the plane of delineation changes lentamente, and this is image shortage contrast as much as to say.Yet, by zeroth order phase place ₀Skew pi/2, image intensity distribute and can be expressed as:

I _image∝sin( ₁- ₀) (52)

The intensity that equation 52 illustrates the present plane of delineation is highstrung around numerical value Δ =0, also presents tangible contrast even be equivalent to this image for adjusting the object phase place purely.

Except improving the intensity contrast, the phase place of skew zeroth order light wave component also can provide the quantitative information about the PHASE DISTRIBUTION of this object.For example, consider the quantity δ that the phase deviation with the zero frequency component can controllably be changed.Any point in the plane of delineation (x, and total electric field E y) (x, y) _ImageAnd intensity I _Image(x, y δ) can represent with following formula, thereby remember that the zeroth order electromagnetic field is constant on the plane of delineation:

I wherein ₀Be the intensity that is associated with the low frequency component, I ₁Be the intensity that is associated with the high-frequency component.

At this, we usually mention the order from the light wave of sample.Yet, when using SLM, in practice, only controllably be offset phase place from the zeroth order component of the light wave of sample and be unusual difficulty.Therefore, in preferred embodiments, our skew comprises the phase place of the low frequency space component of all zeroth order light waves.Therefore, it will be understood that system and method for the present invention can be put into practice by the phase place that only is offset the zeroth order component, and it is optional to be offset the phase place of other order.

By changing δ, can obtain Δ (x, y)= ₁(x, y)- ₀And expression formula:

The phase place that is associated with sample can user's formula 53 and 54 obtain, and these two equations itself use, for example, and total electric field E (x, phase place expression formula y).Represent with following formula with the potential energy mutually that object is associated:

In equation 56, β=I ₁/ I ₀And the ratio of the intensity that is associated with high spatial frequency component and low spatial frequency component respectively of representative.The numerical value of β can be obtained, for example, and the I under four numerical value δ _Image(x, y δ) obtain.

In various embodiment, system and method for the present invention is based on the transmission geometry of microscopic system.Figure 52 is the embodiment that the basis illustrates microscopic system 1200 with the transmission geometry according to the present invention schematically.With reference to Figure 52, a pair of lens, objective lens 1204 and pipe lens 1206 make sample 1210 at plane of delineation P by transmission geometry ₂1212 imagings.Lens L ₃1214 can be used for forming the Fourier transform of image on spatial light wave modulator (SLM) 1216.Central area on the SLM 1216 can be applied to controllable phase deviation δ the central area of incident beam 1220 and reflect whole incident beam 1220 with respect to the remainder of SLM.The central area of incident beam 1220 is corresponding to the low spatial frequency ripple of describing with light beam inner boundary 1222.Outer boundary 1224 illustrates the path of high frequency light beam component, amplifies the dispersing so that in sight of low frequency light beam component and high frequency light beam component.Lens L ₃1214 also can serve as second lens of 4-f system, use spectroscope BS 1232 to form last image on the detector such as charge-coupled device (CCD) 1230.

Device miscellaneous can both be used for controlling SLM and obtain sample image.For example, in various embodiment, thereby the modulation of computing machine 1250 control SLM1216 makes δ increase pi/2 and preferably makes the image acquisition of detector 1230 synchronous.The computing of equation 55 can be finished in real time; Therefore the speed that shows phase image only is subjected to the restriction of the refresh rate of the acquisition time of detector 1230 and SLM1216 in preferred embodiments.

Light illumination mode miscellaneous and lighting source can be used for providing illumination 1260 for transmission geometry of the present invention.This illumination can be finished in bright field pattern or dark field pattern.In addition, the relevant character to used source does not have special requirement.Radiation or " in vain " light (for example, from discharge lamp) that system and method for the present invention can use laser, partly be concerned with.Yet lighting source should have good spatial coherence.

Shown in Figure 52, the component that relevant low frequency and electromagnetic field of high frequency are same light beam; And therefore share altogether to light path.Therefore, low frequency component and high frequency component are influenced by phase noise in a similar fashion, and therefore the various different embodiments of system of the present invention can be counted as the quantitative phasecontrast microscope of no optics noise.For example, in various embodiment, the phse sensitivity of λ in gathering the markers scope arbitrarily/1000 is possible.

In various embodiment, system and method for the present invention is to be suitable for the reflection geometry of microscopic system.Difference between transmission geometry and the reflection geometry is the geometry that throws light on.Transmission geometry can be converted into reflection geometry.

Figure 53 schematically illustrates the embodiment based on the microscopic system 1300 of reflection geometry according to the present invention.The path outer boundary of low frequency component and high frequency component for the sake of clarity is not illustrated in Figure 53.In addition, dispersing for visible of light beam is exaggerated in Figure 53.In various embodiment, spectroscope BS ₁1301 allow second lighting source 1302 to be coupled within this system and provide to throw light on 1303.In one embodiment, second lighting source comprises superluminescent diode (SLD).For fear of because the reflection at various different interfaces causes in light path interference, the low coherence light source such as SLD suits the requirements.

With reference to Figure 53, a pair of lens, objective lens 1304 and pipe lens 1306 make sample 1310 at plane of delineation P ₂1312 imagings.Lens L ₃1314 can be used for making the Fourier transform of image to form on spatial light wave modulator (SLM) 1316.Central area 1317 on the SLM1316 can be applied to controllable phase shift delta the central area 1318 and the whole incident beam 1320 of reflection of incident beam 1320 with respect to the remainder of SLM.The central area 1318 of incident beam 1320 is corresponding to the low spatial frequency ripple.Lens L ₃1314 also can serve as second lens of 4-f system, form last image on the detector 1330 that uses spectroscope BS1332 such as CCD.

Device miscellaneous can be used to control SLM and obtain sample image.For example, in various embodiment, the modulation of computing machine 1350 control SLM1316 makes δ increase pi/2 and preferably makes the image acquisition of detector 1330 synchronous.The computing of equation 55 can be finished in real time; Therefore show that the speed of phase image is limited by the acquisition time of detector 1330 and the refresh rate of SLM1316 only in preferred embodiments.

Reflection geometry also can comprise such as the illumination of using in transmission geometry 1360 according to the preferred embodiments of the invention.Suitable transillumination pattern includes but not limited to bright field and dark field pattern.As according to the present invention in transmission geometry, do not have special requirement about coherence's character of lighting source.System and method of the present invention can use laser, partly relevant radiation or such as " in vain " light of light source from discharge lamp.Yet lighting source should have good spatial coherence.

In reflection geometry, relevant low frequency electromagnetic field also is the component of same light beam with the electromagnetic field of high frequency and therefore shares altogether to light path according to the present invention.Therefore, low frequency component and high frequency component are influenced by phase noise in a similar fashion, and the various different embodiments of system of the present invention can be counted as the quantitative phasecontrast microscope of no optics noise.For example, in various embodiment, the phse sensitivity of λ/1000 all is possible in gathering the markers scope arbitrarily.

In various embodiment, the invention provides the phasecontrast microscope system that utilizes space light wave modulation, this system comprises image-forming assembly and phase imaging assembly.Image-forming assembly and phase imaging assembly energy for example, are built independently, thereby make them be used for existing optical microscope easilier.

Be collectively referred to as Figure 54 A of Figure 54 and 54B and schematically illustrate the embodiment 1400 that the present invention and optical microscope are integrated.The path outer boundary of low frequency component and high frequency component is not for the sake of clarity in Figure 54 illustrated.In addition, dispersing in Figure 54 of light beam is exaggerated for visible.

Phase imaging head 1450 can use, and for example, microscopical video output is docked with optical microscope 1410.Optical microscope 1410 comprises can handle a pair of lens L that light wave makes it the output from sample 1420 to the microscope video ₁1412, L ₂1414 and catoptron 1416.Usually, a part of light wave is guided in order to watch the eyepiece 1426 that light wave is focused on for human eyes 1430 by spectroscope 1424.

Phase imaging head 1450 comprises the lens L that is used for forming the Fourier transform of image on spatial light wave modulator (SLM) 1456 ₃1454.The central area of SLM1456 can be applied to controllable phase shift delta the central area of incident beam 1460 and reflect whole incident beam 1460 with respect to the remainder of SLM.The central area of incident beam 1460 is corresponding to the low spatial frequency ripple.Lens L ₃1454 also serve as second lens of 4-f system, form last image on the detector 1470 that uses spectroscope BS 1472 such as CCD.

The control of SLM and the image acquisition of sample can be done, and for example, use the modulation of control SLM1456 to make δ increase the synchronous computing machine 1480 of image acquisition that pi/2 also preferably makes detector 1470.Computing machine can be a computing machine independently, for example, has the phase imaging head, and perhaps " computing machine " can comprise according to the instruction on the computing machine that the present invention resides in microscope is associated.The computing of equation 55 can be finished in real time; Therefore, the speed of demonstration phase image is limited by the acquisition time of detector 1470 and the refresh rate of SLM1456 only in preferred embodiments.

In various embodiment, the lateral resolution of system of the present invention can improve by the expansion of 4-f system.The 4-f system can be used for transmission geometry and reflection geometry.In addition, the 4-f system can be used to comprise the system of calibration system.Other Fourier operation that the 4-f system makes utilization finish image becomes easy.

Figure 55 schematically illustrates the inventive method and utilizes the system 1500 of 4-f system and an embodiment of method.This 4-f system comprises a pair of lens L ₄1504, L ₅1506, and can comprise spatial filter F 1508.Spatial filter F 1508 provides the amplitude control of indivedual spatial frequencys.With combine by the phase control that SLM provides according to the present invention, the control of described amplitude makes, for example, the cellule device of research cell the inside becomes easily, because the enhancing of high frequency component can improve contrast.People can imagine can preferentially decay other application of some spatial frequency of spatial filter F.

The 4-f system can be added on various transmission geometry embodiment of the present invention and the reflection geometry embodiment.The people that reflection source (for example, second lighting source 1302 among Figure 53) can be familiar with this technology according to the present invention by script at an easy rate uses announcement provided by the invention to be added in the embodiment of Figure 55.

Be used to utilize the system and method for the phasecontrast microscope of space light wave modulation that application miscellaneous is arranged.For example, these system and methods can be used for making micron order and nano level structure imaging.The application of important class is to study iuntercellular and intracellular tissue, dynamics and behavior.By will be altogether being used for the stability that low frequency component and high frequency component provide to light path, and it is all to finish time cycle (from the several hrs to a couple of days) lining research single cell and cell that the ability of measurement makes various preferred embodiment of the present invention be adapted at prolonging by transmission mode and backscattering pattern.Therefore, in various embodiment, the phase imaging that the preferred embodiments of the invention provide is used to provide the information about the slow dynamic process of cell, for example, and size and the change of shape of living cells in the life cycle from mitosis to the cell death.

In various preferred embodiment, method and system of the present invention be used to nano-precision research cell after division detachment process and size, character or both information about cell membrane are provided.The phenomenon that recently has been subjected to special concern is an apoptosis---apoptosis.Suppose that apoptosis can be controlled in the laboratory, in various preferred embodiment, method and system of the present invention is used to study the cytomorphosis of bringing out in this process.In various preferred embodiment, method and system of the present invention is used for studying and survey the difference of the life cycle of various dissimilar cells (for example, cancer cell and normal cell).

People expect that the Fusion of Cells layer has the total interaction to a certain degree that can cause collective's mechanical behavior.In various preferred embodiment, method and system of the present invention is used for studying this total interaction, for example, and by the crosscorrelation between the difference of finishing the phase image that obtains according to the present invention.

The phase imaging that is provided by the preferred embodiments of the invention also can be used to provide the information about the quick dynamic process of cell, for example, and to the reaction that stimulates.For example, the process such as the cell volume adjustment is the reaction that living cells stimulates biological chemistry.The markers of this reaction may from several milliseconds to several minutes and should be able to use the preferred embodiment of system and method for the present invention to measure with considerable accuracy.In various preferred embodiment, method and system of the present invention is used for studying cell to the reaction of biological chemistry stimulation and the engineering properties of measurement eucaryotic cell structure (for example, cytoskeleton).

In various preferred embodiment, method and system of the present invention is used for studying relevant eucaryotic cell structure information, for example understands the transhipment phenomenon of organelle in the cell and creates synthetic biomaterial.In various preferred embodiment, method and system of the present invention is used for studying the cell structure, for example, by use the Mechanical vibration stimulation cell membrane and measure the cell membrane vibration amplitude so that, for example, they and cell engineering properties and cellular material are related.Traditionally, use pearl magnetic or that capture to stimulate this motion.In various preferred embodiment, method and system of the present invention is used for using pearl magnetic or that capture to stimulate the photon pressure of mechanical vibration, femtosecond laser pulse to cause that mechanical stimulus or both study the cell structure.

One class important use is research born of the same parents inner tissue and organelle dynamics.In various preferred embodiment, method and system of the present invention is used for studying the transhipment of various particle in the cell the inside.

Except the diversity of biological study, the preferred embodiments of the invention also are fit to commercial Application, for example, study semi-conductive nanostructured.Semi-conductor industry lacks the reliable quick test of wafer quality in the nanoscale process.In various embodiment, method and system of the present invention is used to provide the nanoscale information about semiconductor structure, for example, and in quantitative mode.In preferred embodiments, nanoscale information has about one second measurement result.

Figure 56 schematically illustrates an embodiment utilizing the phasecontrast microscope system 1600 of space light wave modulation (SLM) according to the present invention.Can use reflection geometry and transmission geometry with 1600 illustrational systems.In addition, this system has the demarcation subsystem.

With reference to Figure 56, a pair of lens, objective lens L ₁1607 and pipe lens L ₂1606 make sample 1610 use catoptron 1613 in plane P 1612 imagings.Imaging can be to use transmission geometry to finish, for example, and by fiber coupler (FC) 1614 with from first optical fiber, 1616 light waves of first lighting source 1620 (being illustrated as He-Ne (HENE) lasing light emitter here) and sample 1610 couplings.Imaging also can use reflection geometry to finish, for example, re-use first spectroscope 1626 guiding on the sample 1610 by FC 1614 and the coupling of second optical fiber 1622 from the light wave of second lighting source 1624 (being illustrated as SLD here) from the light wave of second lighting source 1624.

Lens L ₃1630 are used for forming the Fourier transform of image on the first spatial light wave modulator (SLM) 1632.SLM1632 is used for controllable phase deviation δ is added to the central area of incident beam 1634.In one embodiment, lens L ₃Second lens 1630 that serve as the 4-f system use second spectroscope 1638 to go up at first detector 1636 (being illustrated as CCD here) and form last image.In one embodiment, the system shown in Figure 56 further comprises second 4-f system, and is for example, schematically illustrational in Figure 49.The system of Figure 50 also comprises the demarcation subsystem that is used for demarcating SLM.The nominal light wave trajectory is that with dashed lines 1640 is schematically illustrational, and lighting light wave and imaging wave trajectory are schematically illustrational with solid line 1642.Demarcate subsystem and use a pair of lens L ₄1652 and L ₅1654 (formation optical beam expanders) are collected a part and are transmitted this light wave from the light wave of first spectroscope 1626 and by the polaroid Pc1656 that can be used for switching the SLM operation between phase pattern and amplitude pattern.In order to demarcate, SLM1658 scans the phase deviation of from 0 to 2 π from the beginning to the end by the amplitude pattern, and therefore the phase shift light wave that produces is attenuated when it returns by polaroid.Then, light wave scioptics L ₆1660 are collected and focus on detector 1664.

Multiple device and scheme can be used for controlling the system of Figure 50 and demarcate phase image.In one embodiment, the first control module PC ₁1670 are used to the image acquisition by first detector 1636, and the second control module PC ₂1672 are used for controlling first and second SLM1632,1658 and collect from the data of detector 1664 by oscillograph 1674.Control module PC ₁, PC ₂May be unit or single unit separately.For example, PC ₁And PC ₂May be computing machine or same computing machine separately, described control module can comprise the simulation and/or the digital circuit of the function that is fit to the realization control module.

In various embodiment, system and method for the present invention comprises and for example uses lenticular dynamic focus.In various embodiment, system and method for the present invention comprises parallel focus so that for example, make the two or more imagings simultaneously on the sample.In various embodiment, system and method for the present invention comprises and being suitable for, and for example, visits the coherence function of several points simultaneously aspect the degree of depth.

The present invention utilizes the phasecontrast microscope system of space light wave modulation to operate by two kinds of patterns.In first kind of pattern (after this being called as " amplitude pattern "), obtain Fourier filtering and finish demarcation.In second kind of pattern (after this being called as " phase pattern "), rebuild the wave front of light wave and finish phase imaging.

In various embodiment, in " phase pattern ", do not have polaroid, and light wave is aimed at the fast axis of SLM in the SLM front.Incident light wave is by phase shift, for example, and according to the numerical value that on SLM, addresses.

In " amplitude pattern ", polaroid is placed on the front of SLM.Incident light wave on the SLM by jayrator (as, for example, in " phase pattern " like that), and polarization rotates.When light wave when SLM reflects, it returns by polaroid, and this signal is attenuated.Therefore, aspect amplitude, there is demarcation to reduce based on the SLM phase shift.

Figure 57 A and 57B schematically illustrate the photoelectric effect on the pixel at image, wherein E in amplitude pattern 1700 and phase pattern 1750 _i1702, the 1752nd, the electric field transmitted of incident wavefront, s axle the 1704, the 1754th, the slow axis of SLM, and f axle the 1706, the 1756th, the fast axis of SLM.

Figure 58 A-58C is the block scheme 1800,1850,1855 of the various different embodiments of SLM operator scheme.Figure 58 A illustrates the normal manipulation mode of the setting that is used for phase imaging and describes the SLM operation.RGB 1802 is gray-scale values that control module (for example, computing machine) obtains, and it controls SLM (RGB determines in demarcation to ).RGB is converted into voltage and is used for determining the address of pixel on SLM1804.This voltage is added to, and for example, on the liquid crystal on the SLM, gives incident light wave 1806 phase deviation.Calibration result can obtain in the amplitude pattern.Figure 58 B illustrates the conversion that occurs in the demarcation.Intensity is to use detector 1852 (for example, photodetector) to obtain as the function of gray level image.Then, the function 1854 that is used as gray scale as the intensity of the function of gray level image converts phase place to.Figure 58 C illustrates control-phase pattern and illustrates how calibration result (gray scale) becomes between the actual phase shift that SLM controls and SLM causes to concern in various embodiment.From then on, for example, people can work out the demarcation question blank that is used for this instrument.Be used to produce gray level image 1856 (for example, be used on the computing machine display) as the phase place of the function of gray scale then and be associated with the caused phase shift 1858 of SLM.

Figure 59 is the example to the calibration curve 1900 of the instrument acquisition of operating by the amplitude pattern.Calibration curve 1900 is to be the curve that the phase shift 1902 of unit changes with the gray scale 1904 that is unit with the radian with RGB numerical value.The resulatnt curve 1906 that is obtained is to demarcate the relation between the computer control of inquiring about tableau format displaying SLM and the actual phase shift that SLM causes.Figure 59 can be as demarcating question blank.Burble point 1908 in the curve 1906 is the overlapping of phase place.

Embodiment

Provide some to use the embodiment of transmission geometry to use the embodiment of reflection geometry according to the present invention according to the present invention with some.Appear at, for example, the rotary expansion charactery in Figure 62-66B shows that 2 π blur leveles are eliminated.

Embodiment 1: the phase imaging of calibration sample

In this embodiment, the sample that demarcation is good has been studied and has illustrated the present invention can provide quantitative information with nanometer (nm) scale.Sample is made up of the metal deposit on the glass basis, and is etched then.Shape and metal layer thickness that the metal deposit pattern is numeral " 8 " are the about 140nm that surveys with profilograpy.

Figure 60 A-60D shows the image that the system of use reflection geometry obtains with four kinds of different phase shift delta.Figure 60 A is the image 2000 of δ=0; Figure 60 B is the image 2200 of δ=π; Figure 60 C is the image 2400 of δ=pi/2; And Figure 60 D is the image 2600 of δ=3 pi/2s.

Figure 61 schematically illustrates the high frequency waves vector component E of electric field intensity E 2102 and this electric field _HLow frequency wave vector component E with this electric field _LBetween concern 2100.Shown in Figure 61, Y-axis 2110 and X-axis 2112 are represented the CCD pixel size.Phase place is object " truly " phase place.

Figure 62 is to use the image of the calibration sample 2200 of data (for example, the data of Figure 60 A-60D illustrated) and equation 55 generations.In Figure 62, Y-axis 2202 and X-axis 2204 boths are unit with the pixel on the CCD.Scale 2206 representatives on Figure 62 the right are units of delta with the radian.

Figure 63 is to use the phase image of the sample of demarcating according to system and method for the present invention 2300.Figure 63 is to use equation 56 and data (for example, the illustrational data of Figure 62) to produce.Figure 63 also can user's formula 55 and 56 and data (for example illustrational data of Figure 60 A-60D) produce.Y-axis 2302 and X-axis 2304 are all being that unit and vertical scale 2306 are unit with nm at the CCD pixel.

Shown in Figure 63, people can see that the height of deposit metal pattern 2310 is correctly recovered, provide the ability of quantitative characteristic information to illustrate system and method for the present invention.The noise that exists in the phase image 2300 mainly is to be caused by the inferior quality that is used to write down (8) camera.

Embodiment 2: the phase imaging of phase grating

Nominally nominally Figure 64 shows the phase image 2400 of the phase grating that the dark groove of 10 microns wide and 266nm is arranged that uses that transmission geometry obtains.In Figure 64, Z axle 2402 is a unit with nm, and Y-axis 2404 and X-axis 2406 are unit with the CCD pixel.Vertical surveyors' staff 2408 also to be unit with nm and to determine that according to phase image 2400 degree of depth (Z shaft size) provide in order further helping.

Embodiment 3: the phase image of onion cell

In this embodiment, the onion cell uses the transmission geometry phase imaging according to the present invention.The intensity image 2500 of onion cell is illustrated among Figure 65 so that compare with the phase image of showing in Figure 66 2550.In Figure 65 and Figure 66, y- axle 2502,2552 and x-axle 2504,2554 are unit with the CCD pixel all.Scale 2556 among Figure 66 is a unit with nm.

Intensity image (Figure 65) representative is at first frame that does not have between low frequency component and the high frequency component to obtain under the situation of phase deviation δ=0.Shown in relatively Figure 65 and Figure 66, conventional microscope (intensity) image has low-down contrast with respect to the phase image that obtains according to the present invention.As what in Figure 66, see, improved greatly at the phase image medium contrast, in this case, much meticulous that eucaryotic cell structure can be distinguished.In addition, the information in the phase image is quantitatively arrived the precision of nanometer level and can be converted according to the optical path length of electromagnetic field by this cell.Not only relevant but also relevant with the Nomarski microscope very big improvement of information representative of this type with traditional phase contrast with conventional optical microscope.

The preferred embodiments of the invention comprise that using relevant decomposition that low coherence's optics image field is resolved into two energy controllably is offset the different space component of phase place with exploitation phase imaging instrument with respect to the other side.It is the quantitative phasecontrast microscope of characteristic that this technology is transformed into typical optical microscope with the sensitivity of high accuracy and λ/5500.The result's hint that obtains on the biological cell of living has very big potentiality according to the instrument of the preferred embodiments of the invention for studying biological structure and dynamics quantitatively.

Phase contrast and differential interference contrast (DIC) microscope can provide the hard contrast intensity image of transparent organism structure, need not sample and prepare.The structural information of encoding in phase of light wave is regained by interventional procedures.Yet although two kinds of technology all disclose the sample structure in cross-section (x-y) plane, the information that provides on the longitudinal axis (z) is qualitatively to a great extent.

As this paper in front as described in, the phase-shifting interference measuring method is used considerable time in the quantitative measurement of phase place sample weighing apparatus is learned, and various interfere measurement technique is suggested.Because appearing at phase noise in any interferometer naturally, airwaves and mechanical vibration make the quantitative recovery of the phase place that is associated with optical field become concrete challenge in the practice.Embodiment preferred comprises relevant wavelength so that overcome this obstacle.

In addition, be that cost is recommended the non-interfere measurement technique based on the radiant illumination transport equation with digital computation consuming time for whole audience phase imaging.With the phase image of the modulation of laser emission usage space light wave together with acquisition λ/30 sensitivity.Automatically the digital recording interference microscope (DRIMAPS) of phase shift is to utilize traditional interference microscope that the method for the phase image of biological sample is provided.The measure of the phase noise of the sensitivity of any phase measurement of final restriction though do not employ prevention in DRIMAPS, this instrument application is proved in the potentiality of cell biology.

The preferred embodiments of the invention comprise as the low coherence's phasecontrast microscope (LCPM) that is used for the new instrument of biological study.It is the quantitative phasecontrast microscope of characteristic that this technology is transformed into traditional optical microscope with extraordinary accuracy and extremely low noise.The principle of this technology relies on a relevant electromagnetic field that is associated with optical imagery that decomposes and resolves into its electromagnetic field space average and that spatially change, and these electromagnetism field energys are controllably with respect to the other side's phase shift.Make E that (x is to suppose plural image field static on whole spatial domain y).This field energy is expressed as

E(x，y)＝E ₀+E ₁(x，y) (57)

E wherein ₀Be spatial averaging, E ₁Be the component of E in spatial variations.Therefore, the result of the interference between any image electromagnetic field that can both be regarded as changing on plane wave (average electrical magnetic field) and the space.It should be noted that, as the result of the correct theorem in center, E ₀And E ₁Can in each point of image, be compared to zero of electromagnetic field E-and height-spatial frequency component.So these two space components can fourier decomposition be easy to be separated and carried out phase modulation (PM) independently by finishing.

Experiment package is described in Figure 67.(Axioert35 ZeissC0.) is used for making sample in plane of delineation IP imaging to the upside-down mounting microscope.The low coherence's electromagnetic field that sends by superluminescent diode (centre wavelength in scope 800-850, for example, λ ₀=824nm, bandwidth Delta lambda=21nm, perhaps λ as an alternative ₀=809nn, Δ λ=20nm) are used to transmission beam method.In order to guarantee the total space coherence in power for illumination magnetic field, light wave is coupled among the single-mode fiber and subsequently and collimates with optical fiber collimator.Represent respectively corresponding to electromagnetic field E with dotted line and continuous lines from the light vestige that image displays ₀And E ₁Not deflection light wave and high spatial frequency component.For image field being resolved into the component of describing in the equation 57, fourier transform lens FL (50cm focal length) is placed in apart from the place of plane of delineation IP focal length.People can see in Figure 67, and the MIcrosope image that forms at the IP place seemingly is used as virtual point light source (VPS) illumination of the MIcrosope image of the optical fiber connector that is used to throw light on.So in order to obtain accurate (phase place and amplitude) Fourier transform of image field at the back focal plane of FL, correcting lens CL is placed on planar I P place.The focal length of CL is such, so that VSP is in the unlimited distance imaging; Therefore, the new images of sample keeps its position and enlargement factor, and it is seemingly with the plane wave illumination.In the Fourier plane of FL, kernel frequency components E ₀On optical axis, focus on, and high frequency component E ₁Distribute from axle.In order to control E ₀And E ₁Between phase delay, programmable phase-modulator (PPM) (Hamamatsu Co.) is placed among the Fourier plane.PPM is made up of the two-dimentional liquid crystal array of optics addressing, and it is because birefringence provides accurate control in the phase range of the light wave of its surface reflection.Addressable area minimum on the PPM surface is 20 * 20 μ m ²Or be 26 * 26 μ m as an alternative ², and the dynamic range of phase control is 8 in a wavelength or 2 π scopes.This PPM can depend on the orientation of polaroid P with respect to the liquid crystal main shaft with the phase place (phase pattern of operation) or the amplitude (amplitude pattern) of spatial discrimination mode correction incident electromagnetic field.Back propagated by FL by the light wave that PPM reflects, and on spectroscope BS, be placed in the CCD collection of the conjugate position of IP after the reflection.Therefore, when lacking the PPM modulation, at the accurate phase place of IP place image and amplitude duplicate by the CCD record.The phase place of high spatial frequency component increases continuously by four pi/2 increments, and consequent irradiance distribution can be used the CCD record.Phase modulation (PM) on the PPM and the acquisition rate of CCD are that for example, the computer PC of LabVIEW (American National instrument company) is synchronous by use.The 4-frame phase-shift interferometry of use standard, E ₁And E ₀Between phase difference can be measured.It can be demonstrated the space phase that is associated with image field as important quantity and be distributed with following expression formula.

$\mathrm{\&phi;}(x,y)={\tan }^{-1}\left[\frac{\mathrm{\&beta;}(x,y)\sin \left[\mathrm{\&Delta;\&phi;}(x,y)\right]}{1+\mathrm{\&beta;}(x,y)\cos \left[\mathrm{\&Delta;\&phi;}(x,y)\right]}\right]---\left(58\right)$

In equation 58, factor-beta is represented the amplitude ratio of two electromagnetic field components, β (x, y)=| E ₁(x, y) |/| E ₂|.Parameter beta is measured, thereby the PPM of two spatial frequency components filtering is finished in the same operation with pi/2 popin face (amplitude pattern) selectively.Therefore, user's formula 58, the space phase of given transparent sample distribute and can be regained uniquely.The optimal values of coaxial modulation area is found to be 160 * 160 μ m in the Fourier plane ², and the diffraction spot based on FWHM intensity that is associated with optical system on same plane has the diameter of about 100 μ m.Because the digital computation of equation 58 in fact is instant, so the speed that phase image regains is limited by the refresh rate of PPM only, this refresh rate is 8Hz in one embodiment.Yet total technical speed can other spatial modulator be increased potentially such as ferroelectric liquid crystals by using.

In order to prove the potentiality of its realization quantitative phase imaging, the LCPM technology is applied to studying various standard model.Figure 68 A and 68B show from the example of this measurement result of polystyrene microsphere imaging acquisition.Particle diameter is 3 ± 0.045 μ m that provided by manufacturer (DukeScientific).In order to simulate transparent biological sample better, spheroid is immersed in and is sandwiched in then in 100% glycerine between two cover glasses.The index difference that realizes between particle and surrounding medium is Δ n=0.12.Under the situation that does not have the modulation on the PPM, obtain the typical intensity in transmission image that Figure 68 A shows.People can see the contrast of this width of cloth image because the transparency of sample is very unsatisfactory.Figure 68 B shows the LCPM image that obtains with above-mentioned program outline.Here, the contrast that is obtained comes down to than higher, and the third dimension (Z axle) provides the quantitative information about thickness of sample.Use is described by the profile of the ball centre that Figure 68 B shows, the numerical value that obtains from the diameter of correspondence is 2.97 ± 7.7%, and this is consistent well with the numerical value that manufacturer points out.Existing error may be that the potential impurity that exists in the imperfect and solution of beam quality causes.

The LCPM instrument further is used for forming the phase image of living organism cell.Figure 69 A shows the quantitative phase images of HeLa cancer cell mitosis final stage.It should be noted that cell is surrounded by culture medium, living in before the imaging under the typical condition of culture without any additional preparation.The phase delay that the previous electromagnetic field of having pointed out that the process biological cell is propagated is accumulated and the non-quality of cell are proportional.Therefore, quantitative phase image should such as mitosis, cell growth and dead various stechiology stages of cell kinematics find important use aspect the analysis automatically.

The phase image of whole blood smear is illustrated among Figure 69 B.Sample is to prepare by simply a droplet healthy volunteer fresh whole blood being clipped between two cover glasses.People can see that the well-known oblate shape of red blood cell (RBC) is resumed.Consider that hemochrome is easy to provide quantitative information about cell volume with respect to the simple analysis of the refractive index of blood plasma.Current electronics and the atomic force microscope of only being only applicable to of this level of detail during RBC analyzes.Non-invasive optical technology might provide pathology rapid screening program because well-known be the RBC shape good index of cell health often.In addition, the RBC cell membrane that this can monitor according to the technology of the preferred embodiment of the invention and the complicated dynamic property of peripheral protein matter are the reasons of blood clotting.

For stability and final its sensitivity of quantitative description of assessing instrument antagonism phase noise, make only adorned culture medium (not having cell) the cell container in 100 minutes time cycle by 15 seconds the picture that is partitioned into.Figure 69 C shows the example that rises and falls with the instantaneous phase that is included in certain the spot correlation connection in the visual field.Phase number is at 0.6 * 0.6 μ m ²Area on mean value, it represents the microscopical lateral resolution limit.The standard deviation of these fluctuatings has the numerical value of 0.15nm, is equivalent to λ/5500.The result proves the outstanding sensitivity of LCPM instrument.The extremely low noise that characterizes the instrument characteristic can be with propagate this facts explain in the light path that two relevant electromagnetic fields spatially overlap each other and the phase noise that is subjected to similarly finally to be cancelled influences in interference term.Opposite with laser emission, the use of low coherence's electromagnetic field helps the sensitivity of this method, because the striped that the multipath reflection on various component may form is eliminated.

Therefore, the preferred embodiments of the invention comprise that the sensitivity with high accuracy and λ/5500 levels is low coherence's phasecontrast microscope of feature.Hint the potential structure that is used for the biology system and the valuable instrument of dynamics research of becoming of described apparatus and method about the cancer cell of living and erythrocytic PRELIMINARY RESULTS.By traditional optical microscope is incorporated into this system component, be feature with the multifunctionality of height and easy especially the use according to the instrument of the preferred embodiments of the invention.

Claim should be used as mistakenly is confined to described order or element, unless the sort of effect is had explanation in addition.So, all appear at claims and etc. the embodiment in the scope and spirit scope of value document all in claim scope of the present invention.

Claims (85)

1. method that is used for measuring through the phase place of a part of vectorial light wave, this method comprises the steps:

First wavelength of light wave is provided;

Guide the light wave of first wavelength along first light path and second light path, first light path extends on the medium that will measure, and and second path aspect path, experience change; And

Detection is from vectorial light wave with from the light wave of second light path, so that measure the phase change by the light wave of the point that two are separated on the medium.

2. according to the process of claim 1 wherein that described medium comprises the sample of biological tissue.

3. according to the method for claim 1, further comprise photodiode array is provided at least and with one of fibrous bundle of photodiode coupling so that the phase place of sample in a plurality of locations imaging simultaneously.

4. according to the method for claim 1, further comprise the step of the light wave in frequency displacement second light path.

5. according to the method for claim 1, further comprise the step that changes by at least two photodetector detecting phases.

6. according to the method for claim 1, further comprise the He-Ne Lasers light source that emission first wavelength is provided.

7. according to the method for claim 1, further comprise low-coherence light source is provided.

8. according to the method for claim 1, further comprise first gap and second gap between second reference plane and the 3rd reference plane that are provided between reference plane and the sample.

9. method according to Claim 8, further comprise first reflecting surface that experiences displacement and with originate second reflecting surface of optical coupled of low coherence.

10. according to the method for claim 1, further comprise the step of using polarization discrimination sample gap signal and benchmark event signal.

11., further comprise sample is placed between first reflecting surface and second reflecting surface according to the method for claim 1.

12. a twin-beam measuring system, comprising:

Light source;

The light wave from light source be divided on first light path first component and

The spectroscope of second component on two light paths;

Change the first movable reflecting surface of first optical path length;

Change the second movable reflecting surface of second optical path length;

Guiding the medium that to measure into from the light wave of first light path and second light path

Compositor.

13. according to the system of claim 12, wherein said compositor comprises the light beam spectroscope.

14. according to the system of claim 12, wherein said compositor is the reference field of guiding first reflecting surface that separates by the gap and second reflecting surface from the light wave of first light path and second light path into.

15. according to the system of claim 12, wherein the medium that will measure comprises the tissue that is placed between first reflecting surface and second reflecting surface.

16. according to the system of claim 15, wherein said tissue comprises nerve fiber.

17., comprise that further the light wave that makes from first light path focuses on vectorial first side and makes light wave from second light path focus on lens combination on vectorial second side according to the system of claim 12.

18., further comprise second compositor of light wave being guided into first polarization detector and second polarization detector according to the system of claim 1.

19., wherein be drawn towards the medium and the reference field that the second polarization component is arranged of the first polarization component from the light wave in first path according to the system of claim 1.

20., wherein be drawn towards and medium from the polarization of the light wave quadrature in first path from the light wave in second path according to the system of claim 19.

21., wherein guide the light wave of reference field and the light wave quadrature of guiding reference field from first path into into from second path according to the system of claim 20.

22. according to the system of claim 15, wherein said tissue comprises cancerous tissue.

23. according to the system of claim 12, wherein said light source comprises low-coherence light source.

24., further comprise fiber coupler according to the system of claim 12.

25. a method that is used for measuring the phase propetry of the light wave by a part of sample, this method comprises the steps:

First signal and the secondary signal that are produced by first light source and secondary light source respectively are provided, and secondary light source is a low-coherence light source;

Guide first signal and secondary signal along first light path and second light path;

Change the path length difference between first light path and second light path;

Produce the output signal of indication first and second signals and the optical path delay sum between them;

In interferometer locking modulating frequency modulated output signal; And

Time by the interferometer locking phase changes the phase place of determining with the interactional light wave of sample.

26. according to the method for claim 25, wherein first signal and secondary signal are low coherence's signals.

27., further comprise by using one of frequency mixer or lock-in amplifier demodulation first signal according to the method for claim 25.

28., further comprise with electronics method producing the interferometer locking phase according to the method for claim 25.

29. a system that is used for measuring the phase place of the light wave by a part of sample, comprising:

Produce first light source of first signal;

Interferometer, interferometer produce secondary signal, and secondary signal has because two pulses that the time delay and first signal separate;

From first light path of interferometer and sample relation and second light path of getting in touch from interferometer and reference field; And

Detector system, this detector system are measured respectively from first and second signals of sample and reference field with from first heterodyne signal of the interference between the light wave of sample and reference field reflection; And survey the phase place of indication sample reflection with respect to the heterodyne signal of the phase place of benchmark reflection.

30. according to the system of claim 29, wherein first signal is low coherence's signal.

31. according to the system of claim 29, wherein first light source is one of superluminescent diode and multimode laser diode.

32. according to the system of claim 29, wherein interferometer further comprises first path and second path, there is acousto-optic modulator in second path.

33., further comprise comprising fibre-optic light path according to the system of claim 29.

34., further include low coherence's signal of 5nm bandwidth at least according to the system of claim 29.

35. according to the system of claim 29, wherein said system comprises the heterodyne system Michelson interferometer of shock insulation.

36. according to the system of claim 29, wherein said interferometer further comprises the catoptron of controllably regulating path length difference that is attached on the translation stage.

37. according to the system of claim 29, wherein said detector system comprises first detector and second detector surveyed from the reflected signal of reference field of detection from the reflected signal of sample.

38. one kind is used for making the sample imaging method, this method comprises the steps:

The illumination sample, the light wave that comes from the point on the sample has low frequency space component and high frequency spatial component;

Along to light path high-frequency space component and low frequency space component being concerned with to produce first strength signal altogether;

The phase place of mobile low frequency space component is to produce the first phase shift low frequency space component;

Along to light path the high frequency spatial component and the first phase shift low frequency space component being concerned with to produce second strength signal altogether;

The phase place of mobile low frequency space component is to produce the second phase shift low frequency space component;

Along to light path the high frequency spatial component and the second phase shift low frequency space component being concerned with to produce the 3rd intensity signal altogether;

The phase place of mobile low frequency space component is moved the low frequency space component to produce third phase;

Relevant along making high frequency spatial component and third phase move the low frequency space component to light path altogether to produce top four's degree signal;

At least partially serve as the phase image that the basis produces the point on the sample with first strength signal, second strength signal, the 3rd intensity signal and top four's degree signal.

39. the method according to claim 38 further comprises the steps:

By step to some repetition claims 1 numerous on the sample, thus the phase image of generation sample.

40. according to the method for claim 38, wherein said illumination step comprises uses the transillumination sample that throws light on.

41. according to the method for claim 38, wherein said illumination step comprises uses the indirect illumination sample that throws light on.

42. according to the method for claim 38, wherein said illumination step comprises with transmission and the indirect illumination sample that throws light on.

43. according to the method for claim 38, wherein said illumination step comprises with super illuminating source illumination sample.

44., further comprise the step of the amplitude of controlling at least one high frequency spatial component according to the method for claim 38.

45., further comprise the step of the amplitude of at least one low frequency space component of control and phase shift low frequency space component according to the method for claim 38.

46. according to the method for claim 38, wherein each phase shift step all is offset pi/2 to the phase place of phase change low frequency space component fully.

47. according to the method for claim 38, the step that wherein produces phase image is at least partially based on following equation:

Wherein:

And Iimage (x, y; δ) be point (x, strength signal y), the β=I of the relevant generation of the low frequency space component by high frequency spatial component and phase shift delta on sample surfaces ₁/ I ₀The intensity I that representative is associated with the high frequency spatial component ₁With the intensity I that is associated with the low frequency space component ₀The ratio.

48. according to the method for claim 39, the step of wherein said generation sample phase image comprises the sample phase image of generation phse sensitivity greater than about λ/1000.

49. according to the method for claim 39, wherein said sample comprises biological tissue.

50. according to the method for claim 40, wherein said sample comprises semiconductor wafer.

51. a non-contact optical measurement has the method for the sample of reflecting surface, this method comprises the steps:

First light source that produces first signal is provided;

Use double beam interferometer to produce secondary signal, secondary signal has by the time delay and first signal separates two pulses;

Provide from first light path of interferometer and sample relation and second light path of getting in touch from interferometer and reference field; And

Measure respectively first heterodyne signal from first and second signals of sample and reference field, and from the interference between the reflecting light of sample and reference field; With

Survey the phase place of indication sample reflection with respect to the heterodyne signal of the phase place of reference field reflection.

52. according to the method for claim 51, wherein said first signal is low coherence's signal.

53. according to the method for claim 51, wherein said first light source is one of superluminescent diode and multimode laser diode.

54. according to the method for claim 51, wherein said interferometer further comprises first path and second path, there is acousto-optic modulator in second path.

55., further comprise the light path that comprises optical fiber according to the method for claim 51.

56. according to the method for claim 51, wherein said sample is a part of neurocyte.

57. according to the method for claim 51, wherein said interferometer comprises the heterodyne system Michelson interferometer of shock insulation.

58. according to the method for claim 51, wherein said interferometer further comprises and is attached to the catoptron of controllably regulating path length difference on the translation stage.

59. according to the method for claim 51, wherein said determination step includes first detector and detection detector system from second detector of the signal of reference field reflection of detection from the signal of sample reflection.

60. according to the method for claim 51, wherein said sample comprises biological tissue.

61., further comprise providing and survey the microscope that sample machinery changes according to the method for claim 51.

62. according to the method for claim 61, wherein said sample comprises one of mononeuron and cell monolayer at least.

63. according to the method for claim 61, wherein said microscope comprises the bifocus microscope.

64. a fibre-optical probe that is used for making the sample optical imagery, comprising:

The shell that near-end, far-end and benchmark optical surface are arranged;

Optical fiber in shell near-end and light source coupling; And

At the gradually changed refractive index lens of shell far-end, so that the numerical aperture of described probe provides the effective light wave gathering from the scattering surface of sample.

65., further comprise fibre-optical probe is installed in the system that finishes one of two-dimensional phase imaging and three-dimensional confocal phase imaging on the parallel-moving type objective table at least according to the probe of claim 64.

66. according to the probe of claim 65, wherein said parallel-moving type objective table comprises the scanning piezoelectric transducer.

67. according to the probe of claim 64, the numerical aperture of wherein said probe is in about scope of 0.4 to 0.5.

68. according to the probe of claim 64, wherein said probe makes biological tissue's imaging in vivo.

69. according to the probe of claim 64, wherein said reference surface is the surface on the optical fiber.

70. according to the probe of claim 64, wherein said reference surface is on the gradually changed refractive index lens.

71. a non-contact optical is measured the method for eyes, this method comprises the steps:

The light source that produces first signal and secondary signal is provided;

Provide from first light path of the interferometer of getting in touch with eyes with from second light path of the interferometer of getting in touch with reference field; And

Respond first signal and secondary signal with light wave measurement first heterodyne signal that returns from eyes and reference field respectively; And

Determine light wave that indication returns from eyes phase place with respect to first heterodyne signal of the phase place of the light wave that returns from reference field.

72. according to the method for claim 71, wherein said first signal is low coherence's signal.

73. according to the method for claim 71, wherein said light source is one of superluminescent diode and multimode laser diode.

74. according to the method for claim 71, wherein said interferometer further comprises first path and second path, there is acousto-optic modulator in second path.

75., further comprise comprising fibre-optic light path according to the method for claim 71.

76. a light wave modulating system that is used for making the material imaging, comprising:

Light source;

Lens combination;

The spatial light wave modulator;

The detector of the phase change of detection and the interactional light wave of material.

77. according to the system of claim 76, the diagnostic image of wherein said detector formative tissue.

78. according to the system of claim 76, wherein said lens combination comprises fourier transform lens.

79., further comprise the processor that is connected with detector with the spatial light wave modulator according to the system of claim 76.

80., further comprise low-coherence light source with described system optics coupling according to the system of claim 76.

81., further comprise laser instrument with described system optics coupling according to the system of claim 76.

82. a system that is used for measuring neuron activity, comprising:

Light source;

Make from the light wave of light source and the optical system of the tissue coupling that nerve fibre is arranged; And

Collect from the detector of the light wave of nerve fibre.

83. 2 system according to Claim 8, wherein said system comprise the interferometer of measuring with the phase change of the light wave of tissue interaction.

84. 2 system further comprises the reference field and first and second polarization detectors according to Claim 8.

85. 2 system according to Claim 8, wherein said light source comprises low-coherence light source.

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