CN100380160C - Confocal microscopy imaging system - Google Patents

Abstract

A confocal imaging system utilizing an elongated beam. Specific embodiments are directed to the apparatus with charged couple devices (CCD) and those in which the apparatus is used in fluorescent object observation.

Description

Confocal microscopy imaging system, the method that is used for item inspecting, focus method

Technical field

The present invention relates to a kind of various embodiment that utilize line sweep confocal imaging system and related data handling procedure that comprise, the multiple analysis of the Determination of biological activity by carrying out pair cell extract, cell or tissue, identification is used to diagnose and treat the method and apparatus of the medicament of disease.

Background technology

The method and apparatus of accurately carrying out the analysis of cellular level is current needed aspect drug discovery and development and biological study.The analysis of cellular level helps being used in the biologic activity of assessment compound and the mechanism of action of new biological article.In the analysis of cellular level, the activity of research thing is measured with competition and compensation method.Because for the given activity of compound of great use about the screening of compound, information.For example, not only can assess compound and whether be attached to analyzed target material, actually or and can assess the excitant antagonist of the normal activity of its this target material.Common situation is that this target material is a cell surface receptor.In some signalling channels, the element with maximum potential passage of treatment function is not acceptor but signal protein in the cell relevant with this receptor.Therefore, be necessary to develop a kind of analysis whole passage, the especially method of the activity under cellular environment.

In addition, need the quick and inexpensive a large amount of compound of screening.Thisly need appear at pharmaceuticals industry, usually need test compounds here various biological chemistry target materials, for example activity of acceptor, enzyme and nucleic acid.These compounds are selected from very wide group, approximately surpass 1,000,000 kinds of discrete compounds.Here the purpose of using the term compound for illustrate comprise very wide, the chemical constitution of simple organic and inorganic molecule, protein, peptide, nucleic acid and oligonucleotides, carbohydrates, lipid or any biological study, but be not limited in this.

In the screening compound field, what the analysis of cellular level related to is a pile cell.The reaction of measuring is the average level of whole cell mass normally.For example, one common is used for instrument that ion channel analyzes in Application No. 5,355, open in 215.The typical analysis comprises that the time dependent fluorescence of measuring the ion-sensitive dyestuff, this fluorescence are as being determined at the research ion that adds secondary behind certain compound index in the variation of IC.Dyestuff in the preparation adding cell mass is placed in the bottom, hole of porous plate before mensuration.In general, the reaction of cell all is being different on the time still on the amplitude.This variability may be obstruction or hinder the very observation of important biological concerning screening compound.This inhomogeneity may cause owing to the starting material of experiment, but the more important thing is that inhomogeneity is that any cell mass is intrinsic.Wherein, the origin of variability may be the difference that is secondary to life cycle in the cell mass, or the result of the target material molecule evolution difference of many work.A kind of method of slowing down, compensating even utilize this variation is with the value of enhancing in the cellular level analysis of the feature of compound medicine activity.

The reaction that quantizes individual cells has prevented the heterogencity problem of appearance in the cell mass reaction.Suppose the situation that a very little part reacts to stimulation in cell mass.The device of measuring average response will be lower than the susceptibility of the device of judging the individual cells reaction.A kind of method in back has produced the statistics feature of a reaction overall picture, allows people to go to select from this living cells subgroup.The additional features of cell mass will strengthen the explanation to the reaction overall picture.

The already used various determinators of prior art attempt to satisfy this demand.Flow cytometry analysis is widely used, and measures the characteristic of cell by next laser beam that makes cell pass focusing.But this method has several shortcomings.Pharmaceuticals industry be the most important thing is and can not analyze the compound that is positioned in the microtiter plate easily.In addition, treatment capacity is very low, and typically each sample needs 10-100 time second, is less than 1ms the observing time of each cell, does not allow dynamic analysis, also has, and can only determine the cell average signal.

In addition, a lot of relative positions that need to judge fluorescence signal of analyzing.As Application No. 5,107,422 and the disclosed devices that are known as the scanning cell instrument of Application No. 5,547,849, be widely used in the imaging individual cells.In order to obtain acceptable speed, these devices are in very low resolution (5～10 μ m) operation down.Therefore, these devices analysis need aspect the spatial information that fluorescence signal distributes the comparison type cell instrument by force not what.

Another optional technology is fast picture, whole audience microscope.These devices have the ability that can obtain image under resolution that can compare favourably with the present invention and speed for some sample, yet they are not confocal, therefore are subjected to the influence of background fluorescence easily, and can not be used for the optical cross-section sample.In addition, the data of synchronization gain multiparameter easily.

Summary of the invention

Compared with prior art, the present invention can carry out the multiparameter fluorescence imaging to individual cells or cell mass in enough fast and multiduty modes, is used for screening compound.The method and apparatus that not only is used to obtain and analyze the initial reaction of individual cells but also measures the variability of sample cluster in addition is provided.In addition, also can judge the location of multi-fluorescence with subcellular fraction resolution.At last, the present invention can be used for the incident with the variation of video rate fast imaging.Comprehensive these abilities can make research enter into the frontier of the mechanism of action of drug candidate.

The present invention also can be used in the invention based on the biochemical analysis of fluorescence, and some is similar to the surperficial scintillation analysis (" SSA ") in those methods that are widely used in screening compounds.

Fig. 1 (a)-1 (f) has described the step of receptors bind SSA.At Fig. 1 (a), the dissolvable film 10 with receptor 12 of selecting is added in the hole 20 that comprises liquid 30.These films are separated from the cell of expressed receptor.In figure (b), radiolabeled ligand 14 is added in the hole.Known this part has high-affinity for membrane receptor.Prevailing radioactive label is ³H, ³⁵S, ¹²⁵I, ³³P and ³²P.In Fig. 1 (c), silica bead 16 is added in the hole.These silica bead pan coatings have pair film that the wheat germ agglutinin of strong sticking action is arranged.Silica bead has 3-8 μ m diameter, is made by the plastic material that is doped with scintillator.In addition, the order of the operation of Fig. 1 (b) and 1 (c) description can be exchanged.

Radioactive label is by emission high energy electron or beta particle decay, and this particle is propagated about 1-100 μ m and just stopped according to radioisotopic difference.If this radioactive label is attached on the film attached to the silica bead surface, this beta particle can penetrate the rail pearl and cause luminous.If this radioactive label is dispersed in the liquid, the beta particle of emission generally will not excite silica bead luminous.In Fig. 1 (d), detect the luminous of the silica bead that causes by the radioactive label decay.In Fig. 1 (e), testing compound 18 is added in the hole.The purpose of this analysis is to judge this compound displacement radioactive mark ligand's degree.If radioactive label is replaced and permeated in the liquid, the luminous of silica bead will weaken.In Fig. 1 (f), detect the luminous of silica bead once more.By measuring luminous minimizing, can judge the activity of testing compound.

Fig. 2 (a)-2 (f) has described the step of the receptors bind SSA of another embodiment.This embodiment replaces the silica bead except the embodiment that describes in Fig. 2 (a)-2 (f) uses the material that on the bottom, hole 22 of being made by the plastics of the scintillator that mixed coating can adhesive film, basically with Fig. 1 (a)-1 (f) describe identical.As a result, the embodiment that describes at Fig. 2 (a)-2 (f) detects luminous rather than silica bead luminous of bottom, hole.

Fig. 3 (a)-3 (d) has described the embodiment of an enzyme SSA.In Fig. 3 (a), the silica bead 40 of doping scintillator and the radiolabeled peptide 42 that adheres on it are added in the hole 50 that comprises liquid 60.In Fig. 3 (b), testing compound 44 is added in the hole.In Fig. 3 (c), enzyme 46 is added in the hole.If be not suppressed, enzyme 46 will separate radiolabeled peptides 42 from silica bead 40.As a result, this radioactive label is gone into disperse in the liquid, and radiolabeled decay will not produce luminous on silica bead 40.On the other hand, if these testing compound 44 inhibitory enzymes 46 are typically the active region by sealase, enzyme 46 can not separate this radioactive label, and then radiolabeled decay will produce luminous on silica bead.In Fig. 3 (d), measure the luminous of this silica bead, can determine the activity of testing compound thus.

Fig. 4 (a)-4 (d) has described the embodiment of another enzyme SSA.In Fig. 4 (a), on the bottom, hole 52 of radiolabeled peptides 42 attached to the scintillator that mixed.In Fig. 4 (b), testing compound 44 is added in the hole.In Fig. 4 (c), enzyme 46 is added in the hole.In Fig. 4 (d), measure the luminous of bottom, hole, can determine the activity of testing compound thus.

Top example has been described the rule of SSA, and the activity that promptly is studied thing is analyzed by the variation of the radioactive label number in the cooling length of scintillator.One of attractive force of SSA is not need to remove from the hole in rinsing step attached to the radioactive label on the scintillator.The reason of SSA is similar analysis that Here it is.

Radioimmunoassay (RIA) is the special form of receptor binding assay, and acceptor is a kind of antibody here, and part is modal to be natural or synthetic peptide, protein, carbohydrase or little organic molecule.RIA is a kind of round-about way, is used to be determined at any sample to be tested, and the most common is such as the ligand concentration in the biological specimens such as blood plasma, cerebrospinal fluid, urine or cell extract.At standard RIA, antibody has special affinity for part, and this analysis comprises the radioactive label of antibody, fixed concentration and the unmarked part of unknown concentration.The concentration of unmarked part is by its binding antibody, thereby the degree that has stopped the combination of tagged ligand is judged.RIA be most commonly used to carry out be similar to need with rinsing step will be not the analysis that separates with the part of combination of binding partner.RIA has been developed into the structure of using SSA, and wherein antibody receptor is attached on the scintillator in being doped in silica bead, and rinsing step is omitted.

Yet SSA and RIA also have many deficiencies.At first, these are analyzed needs to handle radioactive material, and this is not only expensive but also time-consuming.The second, these are analyzed only could be effectively in bigger hole.Be directly proportional with the emissivity of beta particle from the luminosity factor of silica bead or hole bottom emission.One typical ³H analyzes to produce and is less than whenever ³The photon that the H decay detects.For increasing the speed of analyzing, must increase radioactive mark ligand's amount, the amount of corresponding film, silica bead and testing compound also will increase.In order to carry out tritium SSA, need to use 10 in 10-60 time second ⁷Individual silica bead.The amount of this silica bead needs the hole of about 150 μ L.SSA is invalid in the hole that is used for screening the required μ L-volume of a large amount of compounds.

The present invention will describe as following with the radioactive mark ligand who uses among fluorescence labeling part replacement SSA and the RIA.Such method, it has been quoted the form of similar RIA and has kept the form of SSA.This can not wash because surface tension makes for the hole particular importance of μ L-volume.Yet with similar forms, the problem as receptor binding assay is described may appear in fluorescence.When adding testing compound, some fluorescence labeling parts are displaced, and disperse is free in the capacity in hole, and other is still attached on the film.This is to be used for judging that the fluorescence of fluorescence labeling part of testing compound activity is attached on the film.But if detect fluorescence from whole hole, the emission that is free on the fluorescence labeling part in the hole will be disturbed the emission attached to the fluorescence labeling part on the film.

Here shown in the problem of mentioning Fig. 5 (a) and 5 (b) in the Application No. 5,355,215 of Schroeder etc.According to the method for Schroeder etc., sample directly is placed on light beam 134 irradiations of bottom, hole in order to the oblique angle, as shown in Figure 5, so it can not shine whole hole.In addition, as light beam irradiates zone 114`, fluorescence has only from being positioned at the pore volume below to be accepted the regional 114a of minimum irradiation and just can detect.

Yet the method for Schroeder etc. still has a lot of deficiencies.At first, because its detects only is sub-fraction at the bottom of the hole, has only the method that on enough big hole, could carry out Schroeder etc. with enough accuracy.It is unsuitable for the sample in the hole of the about 1mm of diameter that imaging is placed on 1536 orifice plates.The second, its geometry has limited light angle, got rid of possibility of the optical module that uses high-NA, and this is imaging micron-scale article, as the individual cells that is positioned at bottom the hole is needed to reach enough light sensitivity and resolution.

Another method that addresses this problem use is the spot scan microscope.For example, in people's such as Baer the Application No. 5,547,849, set forth the use of spot scan confocal system.People's such as Baer method is the slow speed that increases the intrinsic imaging acquisition time of spot scan confocal technology by the sacrifice spatial resolution.For example, people are under certain condition with 10 times of the illuminating bundle enlarged-diameter on the sample, and then the field of illumination increases by 100 times, allow that people are with 100 times faster speed scanning.But the acquisition of this speed be with the loss resolution be cost.In addition, as being disclosed in these pick-up units in the `849 patent, that be fit to described scan method, they are used for the present invention, and then sensitivity is too low.At last, background reflectance degree and resolution is step-down together.Therefore, the device that is disclosed in the `849 patent is compared with the present invention, have lower sensitivity, higher background and lower resolution, and all these is all very important in application now.

The present invention includes novel line sweep confocal microscope.The line sweep confocal microscope is that prior art is known.The J.Microscopy 17117-26 (1993) that two representative enforcement is on the contrary published in the Application No. 5,452,125 of White etc. with by Brakenhoff and Visscher is shown in Figure 7.The both has used a scanning mirror to go to scan illumination by sample.Identical scanning mirror is with scanning (de-scan) fluorescent radiation of making a return journey.After carrying out spatial filtering through a slit, fluorescence group is beneficial to bore hole by scanning once more and observes.The use of vibration mirror makes these microscopes scan vision fast.The line illumination mainly is used in the application that needs fast imaging.The collimation of line illumination the increase compared with an illumination of intrinsic potential speed, only can be simultaneously detect from the light time of each some emission of sample and could realize along illuminating line in imaging system.An essential characteristic of the instrument of the disclosure be have with the plane of object plane conjugation on multiple, detecting element independently.

According to the present invention, sample must be arranged in " plane " of the depth of field decision accurate " planarity " of imaging system.In a preferred embodiment, imaging region is 1mm ²And the depth of field is 10 μ m.Therefore, if whole visual field all will be by synchronizing focus, sample must be 1% smooth.This is correct for the many sample substrates (for example microtiter plate) that are positioned at regional area (as the central area of bottom, hole).Yet it is unpractiaca requiring sample substrates all smooth on whole surface.Because microtiter plate has pact～100mm scope, need 1/10th, 000 planarity.

Optical auto-focusing system provided by the invention can be kept the part " focusing " of sample substrates that will imaging.The optics autofocus mechanism has fast, and the advantage that can for example operate on plastic microtiter and the microslide in nonconductor matrix.Have, this focusing has insignificant operating delay again, that is, the reaction time of focusing is shorter than the Image Acquisition time relatively, preferably part second.The autofocus mechanism based on optics that is suitable for using now is as you know.For example, it is open at Applied Optics 23565-570 (1984) to be used for producing the system based on astigmatic lens that is applicable to servo-controlled positioning error signal, and uses the focus-error detection system of " deflection light beam " open at SPLA200 73-78 (1979).In a preferred embodiment of the invention, sample substrates is a microtiter plate.In this case, the method preferably of finishing focusing more depends on the characteristic of this plate.If the bottom thickness of this plate is a homogeneous, be the sub-fraction of depth of focus, then the retaining plate bottom is having the focusing of a constant deviation just enough with object plane.But the microtiter plate of present common use bottom is not enough homogeneous.Therefore, focusing must be followed the tracks of the resident surface of sample, and this is normally in the hole of microtiter plate.A feature of the present invention is to use the autofocus mechanism of a novelty, is used for focusing on fast noncontinuous surface, as the bottom, hole of microtiter plate.

Therefore, need a kind of method and apparatus to be used for accurately, fast and inexpensive ground, screen a large amount of compounds with similar forms.In addition, also need a kind of method and apparatus to carry out the multiparameter fluorescence imaging with the resolution of enough imaging individual cells and subcellular fraction incident.Also need a kind of imaging system that can go up significant cell mass with additional monitoring and statistics of video rate.

The present invention relates to the line sweep confocal microscope, and line sweep confocal imaging system (LCI) carries out the usage that biologically active is analyzed.

In a preferred embodiment, this line sweep confocal imaging system be used to throw light on sample and fluorescence excitation group sends the multiple wavelength laser light source of electromagnetic energy.These wavelength comprise ultraviolet spectrum and visible light.

The present invention can use the automatic focus performance to carry out a succession of express-analysis on microwell plate, this automatic focus performance allows that this LCI system can promptly move to another hole from a hole, and can not lose the advantage that confocal microscope is differentiated thin this capability of optical cross-section.

In each embodiment of the present invention, mobile sample influences the scanning of the illuminating line on this sample.In a further embodiment, oscillating mirror is used to produce the illuminating line that moves rapidly, and this line influence is deposited in the scanning of the sample of fixed position.Give an example, image can obtain up to the speed of 50 frames with per second.

The present invention preferably provides the distribution of integration, starts the variation of biological event rapidly to allow additional material, as the propagation of the action potential of nerve or muscle cell.

The present invention preferably uses multicomponent solid-state detection device such as charge-coupled device (CCD).This device preferably can be continuous read.At a preferred embodiment, the present invention has used a rectangle CCD, and it has avoided the needs to complete two-dimentional wave detector, allows high reading speed.In addition, can reach big effective field of view at a multilevel scanning embodiment.

The present invention also provides in a preferred embodiment and carried out specific data analysis in image data, thereby allows to handle with a high item performance of handling capacity filtering mode operation.

The invention provides the method for utilizing fluorescence to carry out multiple bioanalysis.In one embodiment, the target material that is studied can be in a fixing cell or living cells, or in subcellular organelle or on the cell membrane.These analyses comprise the judgement to one or more parameters, and this judgement need excite one or more fluorescence labelings, and fluorescence labeling wherein is in general to the different wave length sensitivity of incident light.In addition, these analyses need be with the synchronous and accurate imaging of the light of one or more wavelength emission, and judges position and intensity and their correlativity of fluorescently-labeled sample bidimensional or three-dimensional from this imaging.

In addition, the invention provides a kind of method and carry out such analysis, this analyze to need or relies on the reaction of fluorescent emission and imaging separately, or the quick repetition imaging of same zone or cell.In each embodiment, imaging is carried out with the speed of per second 50 frames.Under the multi-wavelength situation, with high spatial resolution apace this ability of imaging allow the present invention to carry out the analysis that can not be carried out in advance, perhaps carry out these analyses in a kind of senior mode.

The present invention relates to be used for screening compounds, and be used for the various several methods that involve the polytype analysis of using fluorophor or fluorescence probe.In general, these analyses and screening sequence comprise the use of testing compound and reagent, and some or all in them are primary fluorescences, become fluorescent product with fluorescence labeling or metabolism.Testing compound and reagent can combined in various manners.

In one embodiment, reagent is added in the hole that comprises liquid.This can be one of them hole in many holes on a single hole or the porous plate.The biologic activity that is studied material is being positioned at the bottom, hole or is existing on the silica bead surface of bottom, hole or do not exist fluorescence group to determine by what measured by the line sweep confocal microscope.This embodiment is the same with the SSA form, promptly judges activity from the localization of the sample that detects.Under the SSA situation, localization approaches the center of scintillator.In the method, the location is the zone in a hole, is preferably in the bottom.In SSA, the susceptibility of contiguous sample is determined by the attenuation length of beta particle.In the method, determine by the optical cross-section degree of depth of confocal microscope for the susceptibility of the fluorescence of localization.

In addition, the present invention can mode fast and automatically carry out the high transaction throughput analysis that needs a plurality of samples of screening.These samples can be independent micropores, maybe can be the holes that includes liquid, living cells, fixed cell or cell component.The present invention also is provided at the environment contrast that needs the liquid hold-up sample during the analysis or keep activity of living cells.

Other purpose of the present invention, feature and advantage will become clearly from following writing up.

Description of drawings

Fig. 1 (a)-1 (f) shows the SSA of first receptors bind.

Fig. 2 (a)-2 (f) shows the SSA of second receptors bind.

Fig. 3 (a)-3 (f) shows the SSA of first enzyme.

Fig. 4 (a)-4 (f) shows the SSA of second enzyme.

Fig. 5 (a) and 5 (b) are used for the skeleton diagram of equipment that imaging is positioned over the prior art of the sample at the bottom of the hole.

Fig. 6 is the skeleton diagram according to first embodiment of the line sweep confocal microscope that is used for the imaging sample of the present invention.

Fig. 7 is the microscopical skeleton diagram of prior art.

Fig. 8 (a) and 8 (b) are respectively the top view and the side views of the raypath of the colored embodiment that does not have a scanning mirror of the present invention.Fig. 8 (c) is the top view of the self-focusing raypath of single bundle.

Fig. 9 (a) and 9 (b) are respectively the top view and the side views of the raypath of the colored embodiment with scanning mirror of the present invention.Fig. 9 (c) is the top view of the self-focusing raypath of single bundle.

Figure 10 is the side view of two bundle autofocus systems.

Figure 11 (a)-11 (c) shows rectangle CCD camera and readout register.

Figure 12 (a) and 12 (b) are the sectional views that line sweep confocal microscope of the present invention uses the raypath of traditional dark field imaging formation.

Figure 13 (a) and 13 (b) are the sectional views that line sweep confocal microscope of the present invention uses the raypath of being inverted dark field imaging formation.

Figure 14 is the sectional view that line sweep confocal microscope of the present invention uses the raypath of being inverted dark field imaging formation, and the zone here is greater than the diffraction limit zone of illuminated sample plane.

Figure 15 (a)-15 (f) shows first embodiment according to receptor binding assay of the present invention.

Figure 16 (a)-16 (f) shows second embodiment according to receptor binding assay of the present invention.

Figure 17 (a)-17 (f) shows first embodiment that analyzes according to enzyme of the present invention.

Figure 18 (a)-18 (f) shows second embodiment that analyzes according to enzyme of the present invention.

Figure 19 (a)-19 (p) shows the reaction of neuroblastoma metabolic defect in cellular calcium ion to carbaminoylcholine.Show respectively in the preform injection time, 0,1.2,2.4,3.6,4.8,6.0,7.2,8.4,9.6,10.8,12.0,13.2,14.4,15.6 seconds reaction (that is Figure 19 (a)-(o))

Figure 20 (a)-20 (h) shows the reaction of neuroblastoma metabolic defect in cellular calcium ion to the KCl of 50mM.Show respectively 0,0.6,3.0,5.4,7.8,10.2,12.6 and 15.0 seconds reaction (that is Figure 20 (a)-(h))

Figure 21 (a)-21 (c) shows living cells receptor binding assay of the same race.Figure 21 (a) shows the cell of fluorescein-labeled peptide part dyeing.Figure 21 (b) shows the mask that tenuigenin produces with LDS 751 dyeing.Figure 21 (c) is the overlapping of Figure 21 (a) and 21 (b), and it has shown variable receptor active.

Figure 22 (a)-22 (d) shows living cells receptor binding assay of the same race.Figure 22 (a) be mark with the diagram of unlabelled part competitiveness in conjunction with cell surface receptor.Figure 22 (b)-22 (d) is presented at the image of the Immunofluorescence Reactions of table middle finger anchor point.

Figure 23 (a)-23 (d) shows the living cells receptor binding assay of the same race of Cy3 tagged ligand.

Figure 24 (a)-24 (d) shows 4 μ m diameter silica beads with different number Cy5 marks.Figure 24 (a) has shown the image of the silica bead that does not have mark.Figure 24 (b)-(d) has shown the image of the silica bead of every pearl average 17,170 or 1700Cy5 labeled molecule respectively.The image that Figure 24 (d) shows has dwindled 10 times than the picture size of Figure 24 (a)-24 (c).

Figure 25 (a)-25 (c) shows the data of analyzing as the ion channel in the table.Figure 25 (a) relates to the reaction of neuroblast oncocyte and additional carbaminoylcholine.Figure 25 (b)-(c) is shown as the reaction of nerve-cell tumor and the calcium ion of additional 50mM KCl.

Embodiment

The integral body that is listed in here all patented claims, publication and other reference material is cited as a reference.

The present invention is used for the used medicament of identifying disease treatment.Here provide a kind of one or more fluorescent reagents that use to measure the method that high-throughput biological respinse, that can carry out multiple bioanalysis is handled item.This analysis can be at chemical mixture or any biopreparate molecule that is studied, and comprises but be not limited only to for example those drug test product of finding in combinatorial libraries.In addition, the invention provides a kind of being used for from the method for cell and tissue samples diagnosis pathological state.The present invention also provides a kind of and is used to use fluorescent reagent to show the method for drug test product to the multiple biological respinse of whole cell.

Technology of the present invention can be used in analysis with enough fast speed from data individual cells, cell or subcellsular level, thereby allow in the cell mass sample that constitutes statistical significance of q.s and to obtain this data.The present invention can carry out multiparameter and measure synchronously, and makes a plurality of signal corrections from individual cells.Therefore it can be used in and analyzes heterogeneous cell effect, and the reaction of analyzing the inferior collection of cellule that limits.

In addition, the synchronous activation that the present invention can imaging multiple signal path, and make these multiple signals relevant simultaneously or relevant in time.This performance seems very important when a specific analysis needs the transient response of individual cells of the transient response of individual cells or contrast.

In addition, can there be unconjugated fluorescence group in the present invention or at compound, comprises when having primary fluorescence in the potential drug test product the confocal planar imaging fluorescence signal from cell.

These analyses can be used any known fluorophore or fluorescence labeling, comprise dyestuff Cy3, Cy5, Cy5.5 and the Cy7 of fluorescein, rhodamine, Texas Red, Amersham Corp., the nuclear dyestuff nuclear Coumarin dyestuff of Hoechst.(see Haugland, R.P. fluorescence probe and the chemical handbook of research, sixth version, 1996, molecular probe, Inc., Eugene, Oregon.).

These analyses include but are not limited to this: receptor binding assay, intracellular potential or pH analysis, ion concentration analysis, enzyme activity assay, traffic analysis, dynamic imaging analysis and precious cell event analysis.

Receptors bind and enzyme activity assay can be based on silica bead or based on the analysis of cell.Some examples based on silica bead are described in WO98/55866.Yet what the method that the there is described was used is the spot scan confocal technology, and the line sweep confocal imaging system that the present invention uses has significant advantage aspect data collection rate.

Optical texture

Fig. 6 shows the first embodiment of the present invention.This microscope for example comprises at light penetrates electromagnetic radiation source 400 or 410 in the 350-750nm, cylindrical lens 420, primary fissure pore membrane 430, first relay lens 440, dichronic mirror 450, object lens 470, the microtiter plate 480 that comprises the sample aperture 482 of two-dimensional array, pipe lens 490, filter 500, secondary fissure pore membrane 510 and detecting device 520.These elements are arranged along optical axis OA, aperture, crack 432,512 on the slit membrane 430,510 and the plane vertical stretching of Fig. 6.Lens 440,470 and 490 focal length and the distance between the distance between these lens and slit membrane 430 and the lens 440, the distance between object lens 470 and microtiter plate 480 and lens 490 and the slit membrane 510 equates, a confocal microscope so is provided.In this embodiment, it is in alignment to use cylindrical lens 420 to focus on the electromagnetic radiation of sending from lamp 400 or laser instrument 410.The shape of this line is optimized through primary fissure pore membrane 430.Slit membrane 430 is described on the plane of delineation of optical system, this plane and object plane conjugation.The illumination band scioptics 440, dichronic mirror 450 and the object lens 470 that are formed by the aperture on the slit membrane 430 432 are relayed to the microtitration orifice plate 480 that comprises two-dimensional array sample aperture 482.For convenience of description, the optical element of Fig. 6 is with section form, but orifice plate is described with perspective form.The projection of illuminating ray on orifice plate 480 formed by line 484, is appreciated that it is equally perpendicular to the plane of Fig. 6.As arrow A and B indication, microtitration orifice plate 480 can (X, Y) direction moves in two dimension in the direction that is parallel to the array (not shown).

At another embodiment, slit membrane 430 is positioned at Fourier (Fourier) plane of optical system, promptly with the plane of back focal plane (BFP) 460 conjugation of object lens.In this case, aperture 432 is arranged on the plane of figure, and lens 440 are relayed the illumination band that formed by aperture 432 on the back focal plane 460 of object lens 470, and its changes light becomes perpendicular to the line 484 on the object plane on the plane of Fig. 6.

In another embodiment, this slit membrane 430 is removed fully.According to this embodiment, lighting source is a laser instrument 410, and the light that sends from laser instrument is focused the back focal plane 460 of object lens 470.This can be by as shown in Figure 6 cylindrical lens 420 and the combination of spherical lens 440, maybe can 460 finish to the plane with the direct focused ray of cylindrical lens.

To sample plane, fluorescent emission reflection there is to detecting device 520 by projection lighting light, at direction movable plate 480 perpendicular to light, can with detecting device 520 read synchronization gain sample areas, for example image of the sample in sample aperture 482.In the embodiment that Fig. 6 describes, fluorescent emission is collected by object lens 470, through double-colored spectroscope 450 projections, pass filter 500 and secondary fissure pore membrane 510 and be transmitted on the detecting device 520 by another object lens 490, be suitable for having the confocal imaging system that object lens 470 are proofreaied and correct in the infinite distance like this.Double-colored spectroscope 450 and filter 500 preferentially are blocked in the light in the illumination wavelengths.Detecting device 520 is actually a camera, can be one dimension or bidimensional.If what use is one-dimensional detector, then do not need slit membrane 510.Before the appointed area was by imaging, illumination, detection and conversion were carried out continuously.If sample with continuous rate transition mechanical motion can simplify.If the camera time for reading is less than the time shutter, then continuous motion is the most useful.In a preferred embodiment, camera is read continuously.In time shutter that merges and time for reading, the displacement d of sample can be greater than or less than the illuminating line width W, for example 0.5W≤d≤5W.The mode imaging that all holes can be same on the porous plate.

In addition, microscope can be configured to focus illumination light and pass many holes adjacent, that mainly limited by the visual field of optical system, and the result can use more than one microscope simultaneously.

The size of illumination band 484 and shape are determined by the width and the length of the Fourier transform band of objective lens ' 460.For example, the length of line 484 is determined that by 460 width of reaching the standard grade on the contrary, 484 width is determined by 460 length.For diffraction limit performance, select length at the illumination band at 460 places to be full of aperture, object lens rear portion.Those skilled in the art are readily appreciated that, the size of illumination band 484 and shape can promptly be controlled by the aberration of the effective numerical aperture on each size, object lens and the restriction in the object lens visual field by the combination control of the focal length of cylindrical lens 420 and 420 places bundle spot size.

The size of selecting illuminating line 484 is to optimize signal to noise ratio (S/N ratio).As a result, they are that sample relies on.According to this analysis, resolution changes between diffraction limit,, is less than 0.5 μ m that is, is approximately 5 μ m.The length of bundle spot preferably determined by the visual field of object lens, for example 0.5 and 1.5mm between.For example, Nikon ELWD, 0.6NA, the 40X object lens have the visual field of about 0.75mm.The diffraction limit resolution of the 633nm radiation of this object lens approximately is 0.6 or is approximately 1100 resolution elements.

Significant depth resolution is mainly determined by the aperture width on the slit membrane 510 512 or one-dimensional detector width with by the image enlargement factor that object lens 470 and lens 490 are united generation.The high depth resolution of confocal microscope is near 1 μ m.In this application, the depth resolution that 5-10 μ m is high is enough very good in other words.

For example, when the sample that is studied, as living cells, the fluorophor that comprises in the diffraction limit volume was not enough to allow obtain the satisfactory SN ratio image in the enough short image acquisition time, irradiation and collecting from being favourable than the emission the bulky volume of diffraction limit.With video rate dynamic studies transient affair, for example be more suitable in this way under the similar situation that ion channel is opened.In fact, this can be by being full of the aperture, rear portion of object lens, and the method that is equal to the diameter that increases illumination aperture is finished.The effective numerical aperture (" NA) of illumination is less than the NA of object lens.Yet fluorescent emission is collected with the full NA of object lens.The width in aperture 512 must increase, to detect the emission of bigger illumination volume.During greater than several times of diffraction limits, geometrical optics provides enough approximate value for the size of detection volume unit in aperture width.

Lateral depth: a _d=d _d/ M

Axial depth: $z_d=\sqrt{{2a}_d}/\tan \mathrm{\α}$

Here, M is an enlargement factor, d _dBe the width in aperture 512, α is the half-angle that object lens 470 are faced toward.One of pith of the present invention be exactly among the embodiment illumination aperture 432 or its equivalent when having aperture and detection aperture 512, can not controlled separately.

The multi-wavelength structure

The embodiment that enables the multi-wavelength fluorescence imaging is more suitable to some analysis type.Because in the biological respinse field time is a key factor, so two kinds or more of measuring method is carried out simultaneously, in general is favourable, and is necessary.

The quantity of individual wavelengths or color is relevant with the concrete analysis of execution.In one embodiment, three illumination wavelengths have been used.Fig. 8 (a) and 8 (b) illustrate the raypath in three looks (three-color) the line sweep confocal imaging system respectively from top view and side view.In a word, this system comprises several electromagnetic radiation source S _n, calibration lens L _nWith mirror M _n, this mirror is used for producing by cylindrical lens CL focusing the becoming first spatial filter SF ₁On the collimated beam spot of fasciculi exilis spot, be positioned at the first spatial filter SF ₁With the second spatial filter SF ₂With imaging len IL, beam splitter DM ₁And DM ₂And the confocal microscope between the detecting device D, be used for separating and detecting the different wave length composition of fluorescent radiation from sample.Spatial filter SF, SF ₁And SF ₂Slit membrane preferably.

Particularly, Fig. 8 (a) has described about color λ ₁, λ ₂And λ ₃Light source S ₁, S ₂And S ₃, and calibration is from the lens L of the light of light source separately ₁, L ₂And L ₃Lens L ₁, L ₂And L ₃Preferably be adjusted to any dyeing of other lens that can compensate in this system.Mirror M ₁, M ₂And M ₃Be used for combination from light source S _nLighting colour.Mirror M ₂And M ₁Part transmission, partial reflection and preferably double-colored.

The operation microscope need be from light source S under confocal pattern _nThe excitation source through combination be focused into " line " on object plane OP, perhaps high eccentric elliptic.In conjunction with the discussion that Fig. 6 did, can use multiple configuration to realize this function as above.In the described embodiment of Fig. 8, the lighting source through making up focuses on the ellipse that is elongated by cylindrical lens CL, this ellipse and spatial filter SF ₁In the slit unanimity.Drawn in Fig. 8 a and 8b, slit membrane SF ₁Be placed in the plane of delineation of this system, put the direction of propagation, and its major axis is in the page of Fig. 8 a perpendicular to illuminating ray.Lens CL and OL relaying are from comprising SF ₁Illuminating ray to object plane OP.The effect of rotating mirror TM is convenient the use.In another embodiment, DM ₃Between TL and OL, CL directly focuses on BFP to illuminating ray.For those skilled in the art, other embodiment is obvious.

With reference to figure 8 (b), send and be imaged onto spatial filter SF by tube lens TL by the light that object lens OL assembles by sample ₂On.SF ₂Preferably put the seam that extends perpendicular to the page or leaf plane.Therefore, by light filter SF ₂Light be an illuminating line basically.SF ₂Can be placed on the conjugate planes initial imaging plane or any there.DM ₃Be partial reflection, the part transmission, preferably colored.Can obtain preferably can reflecting the interior wavelength of certain wave band and multi-wavelength " double-colored " mirror of its commplementary wave length of transmission, or " colour " mirror.

δ λ ₁Be defined as by λ ₁The excited fluorescent radiation.In general, this is to compare λ ₁Chang Wavelength distribution slightly.δ λ ₂With δ λ ₃Definition similar.DM ₃Preferential reflection λ _n, preferential transmission δ λ _n, n=1,2,3.By SF ₂The light of transmission is imaged on the pick-up unit, and this pick-up unit is positioned on the plane with initial imaging plane conjugation.In Fig. 8 (a), spatial filter SF ₂Image be to result from all three detecting devices, D by lens IL _nOn.This embodiment be needs almost completely be recorded in the application that produces by each self-detector between the image preferably to come out.In another embodiment, each lens IL _nLink to each other with pick-up unit, a pair of lens IL and ILn are as relaying spatial light filter SF ₂Image to each detecting device D _nLight in detecting device by mirror DM ₁And DM ₂Separate.Mirror is part transmission, partial reflection, preferably double-colored.DM ₂Preferential reflection δ λ ₂And preferential transmission δ λ ₃Barrier filter BF2 and BF3 be preferential transmission δ λ respectively ₂With δ λ ₃, effectively stop other wavelength that is occurred.

The scanning mirror structure

In some embodiments of the invention, high-speed data acquisition need be in the video rate viewfinder image.General per second 30 to 60 frames of video rate imaging.In present application, the intention hint has the frame speed of the 30Hz order of magnitude.In a preferred embodiment, by one dimension illumination, and,, realize the video rate imaging so that cause the relative displacement of illumination and sample by scanning perpendicular to the primary beam spot on its direction along sample plane.In general this scanning stage is measured very big.Therefore, it can not move rapidly fully.

Fig. 9 has described one embodiment of the present of invention of using scanning mirror SM.This mirror is fit to be placed on that (obiective back focal plane, BFP) on conjugation plane: the rotation in this BFP plane of its conjugation (or with) lining causes the displacement in this object plane (OP) and its conjugate planes with objective lens '.For lens RL ₁And RL ₂The representative value of focusing length, the whole sweep limit of SM only needs the several years.As shown in Figure 9, these a pair of lens are BFP 1 to be imaged onto on the SM with magnification, but can use various magnifications easily.The limiting factor of image acquisition speed is the reading speed and the signal intensity of camera.In imaging pattern described above, data can be with the reading speed of camera, and for example 1MHz obtains continuously.The most handy scanning mirror uniaxially obtains data.It is that sawtooth moves that the ideal scan that allows people to obtain data is continuously moved.In practice, turn back and the combination of sweep time of returning will constitute～a 1/3-2/3 scan period.Suppose that be 50% idle time, then the pixel pick-up rate of the mirror vibration frequency of 50Hz and 1MHz～10,000 pixels will obtain each frame with per second 50 frames, and this enough is used for, and a frame one frame ground is differentiated and the individual articles of tracking such as cell.But each image 10 ⁴Individual pixel lacks 10 than the generalized case of above consideration ²Doubly.According to this application, its advantage is to obtain relatively little image under high resolving power, and under lower resolution, obtain relatively large image, for example pixel when being 0.5 μ m * 0.5 μ m image be 50 μ m * 50 μ m, and image is 200 μ m * 200 μ m when pixel is 2 μ m.

Automatic focus

According to the present invention, sample must be positioned at the object plane of imaging system.Therefore, the invention provides a kind of autofocus mechanism, it maintains the sample portion in the visual field of this imaging system within the object plane of that system.The degree of accuracy of complanation is determined by the depth of field of this system.In a preferred embodiment, the depth of field approximates 10 μ m, and the visual field approximates 1mm ²

Disclosed autofocus system is with negligible deferred run, promptly its response time shorter with respect to the acquisition time of this image, such as 0.01-0.1s.In addition, this automatic focus light source is independent of lighting source and sample properties.Especially this configuration allows the position of this sample container to place along the definite optical axis in position that imaging system will be independent of object plane.

Fig. 8 and 9 provides single bundle a self-focusing embodiment, and wherein showing wavelength is λ ₄Independent light source S ₄With detecting device D ₄Wavelength is λ ₄Need be different from sample fluorescence, particularly the wavelength that can not excite the tangible fluorescence in the sample.Therefore, λ ₄Being preferably near infrared light, for example is 800-1000nm.Part transmission, partially reflecting mirror DM ₄Preferably dichromatic, reflection λ ₄With transmission δ λ _n, n=1,2,3.Be applicable to that the autofocus mechanism based on optics of the present invention is known.For example be used to produce be suitable for servo-controlled position error signal, be disclosed in Applied Optics 23565-570 (1984) based on the system of astigmat.Use the focus-error detection system of " asymmetric bundle " to be disclosed in SPIE 20073-78 (1979).The latter's method realizes D wherein according to Fig. 8 and 9 easily ₄Be separate detector.

Have the microtiter plate that is placed with sample at the bottom of the hole in order to use, servo loop must interrupt, so that move between the hole.This can cause sizable time delay, and reason is to throw light on each time all to need when being moved to another hole to focus on again.

In preferred embodiment shown in Figure 10 of the present invention, provide the continuous closed-loop control of the relative position of sample plane and object plane.This system uses two independently electromagnetic radiation beams.Focus on continuous level from that of S5, for example at the end of microtiter plate.From S ₄That focus on discontinuous plane, for example at the bottom of the hole of microtiter plate on.In one embodiment, from S ₄And S ₅The bundle spot have wavelength X respectively ₄And λ ₅λ ₄By L ₄Calibration is by aperture I ₄Form the hole, focus on discontinuous surface by object lens OL.λ ₅By L ₅Calibration is by aperture I ₅Form the hole, focus on continuous surface jointly by object lens CFL and object lens OL.The light of reflection is respectively by lens IL ₄And IL ₅Focus on detecting device D ₄And D ₅The part transmission, partially reflecting mirror DM ₄Best dichromatic reflection λ ₄And λ ₅, transmission λ _nWith δ λ _n,=1,2,3.Mirror M ₄, M ₅And M ₆Be the part transmission, partial reflection.At λ ₄And λ ₅Not not simultaneously, M ₆Preferably dichromatic.

According to this embodiment, wherein sample resides in the microtiter plate, λ ₄Be focused the bottom in hole.Object plane can be in bottom, variable range bias internal hole.This can be by adjusting L4 or finishing by the skew adjustment in the servo control loop.For convenience of description, can suppose λ ₄Focus on object plane.

The operation of autofocus system is described below.If the bottom of sample aperture is in the focal plane of object lens OL, detecting device D ₄Produce an error signal that offers Z control by switch SW.Z control is used for the motor (not shown) of mobile microtiter plate toward or away from object lens.In addition, Z control can mobile object lens.If the bottom PB of microtiter plate is not at the focal plane of lens CFL and object lens OL combination, detecting device D ₅Produce an error signal that offers Z control by switch SW.An XY control control is used for the motor (not shown) of mobile microtiter plate to the object plane OP of object lens OL.

As indication, whole scanning is carried out under computer control.Below for the example of a scanning: after the imaging in a specific hole is finished, computer run SW with the control of servo control mechanism from by D ₄The error signal that produces switches to D ₅The error signal that produces.Computer instruction XY control goes movable plate to arrive next hole then, the servo afterwards D that switches back again ₄.

Utilize the hole of position in the thickness of plate bottom that " slightly " focusing of the signal of plate bottom is used to keep sample plane to pitch-row from interior, thereby the scope that needs " carefully " focusing to search for is reduced.For example, the diameter of aperture I5 is 2mm, IL ₅Be 100mm, then the picture size on detecting device will be～100 μ m.Equally, if aperture I ₄Diameter is 0.5mm, IL ₄Be 100mm, then the picture size on detecting device will be～400m.The latter is chosen to muting sensitivity so that it plays " slightly " focussing force.

As the embodiment that contains list-bundle spot described above, wavelength X ₄And λ ₅Need be different from sample fluorescence, particularly can not excite the wavelength of the obvious fluorescence in the sample.Therefore, λ ₄And λ ₅, for example be 800-1000nm preferably near infrared ray.In addition, these two wavelength are preferably distinguishing, for example λ ₄=830nm, λ ₅=980nm.

In self-focusing another embodiment of two-bundle spot, λ ₄=λ ₅And this two bundles spot can be from same source.Best this two bundles spot is polarized vertical mutually and M6 is polarization beam apparatus (beamsplitter).

In the self-focusing preferred embodiment of single bundle of operation in the following manner, provide pseudo-closed-loop control.Move to SW at described plate and be switched back thereafter D ₄Next hole the time, computing machine switches the sample stationary installation to control at the end of single pass operation SW, this device is kept Z control output a constant level, this moment, plate was moved to next hole, SW is switched back D afterwards ₄

Pick-up unit

The essential characteristic of equipment disclosed herein be with the plane of object plane conjugation in, used pick-up unit with multiple independently detecting element.As previously discussed, the advantage of line illumination need in the application of rapid imaging mainly to be.Compare with a lighting condition, potential speed increase is the collimation inherent characteristics of line illumination, yet has only this imaging system to have the ability to detect simultaneously along the light of illuminating line emission from the every bit of sample, could realize.

Imaging system (White etc. in prior art described above, US 5,452,125 and Brakenhoff and Visscher, J.Microscopy 17117-26 (1993)) charge-coupled device (CCD) (CCD) is installed in output place or other camera also is fine.But the equipment of Gou Chenging has been compared three significant disadvantage with the present invention like this.One is to rescan into image on the two-dimensional detector, and this has increased equipment is unnecessary complexity.Another is the enough high-quality full two-dimensional detector that needs 1000 pixels * 1000 pixels that surpass general architecture camera.The 3rd shortcoming is to need the extra time to read entire image from two-dimensional device.

The present invention is designed to avoid these shortcomings, and not only optimizes image taking speed under the restrictive condition of high sensitivity and low noise detection, and optimizes handling capacity.The line camera that embodiment uses to read continuously, and in a preferred embodiment a rectangle CCD is used as the line camera.Between the line of these two embodiment in an image or all do not have idle time between the image.An attendant advantages of the present invention is: can obtain bigger effective field of view in stage scanning embodiment, this will be in following discussion.

The necessary characteristic of this pick-up unit can further be elaborated by considering following preferred embodiment.The resolution limit of object lens is＜1 μ m, is generally～0.5 μ m, and detecting device comprises the array of being made up of～1000 independent components.Resolution, the visual field (FOV) and image acquisition speed are not independent variables, need coordinate in these performance parameters.Generally speaking, the magnification of this optical system is arranged under the situation of not sacrificing resolution, makes image the same with FOV as far as possible big.For example ,～visual field of 1mm can be imaged on the pixel of 1 μ m on 1000 element arrays.If detecting element is 20 μ m squares, then the magnification of system will be arranged to 20X.Notice that this can not produce 1 μ m resolution.Pixel is not equal to resolution.For example, if the intrinsic resolution limit of object lens is 0.5 μ m, and each the 0.5 μ m in the object plane * 0.5 μ m zone is mapped to a pixel, and then the true resolution of the digital picture of Chan Shenging is not 0.5 μ m.In order to obtain true 0.5 μ m resolution, this pixel must be corresponding with one the 0.2 μ m * 0.2 μ m zone in the object plane.In one embodiment, the magnification of imaging system is arranged to obtain real optical resolution.

At present, have the highest detection efficient of the enough reading speeds that are suitable for the application, the pick-up unit of lowest noise is the CCD camera.Figure 11 illustrates a rectangle CCD camera with mxn array of detector elements, wherein the m strictness is less than n.The image that fluorescence sends has covered and has been close to the delegation that reads register best.This makes the transmission time minimum, and has avoided the illuminated mantissa of going and reading in the row between the register is added in the signal.

In theory, people can be arranged so that slit SF on the CCD camera to the magnification of optical system ₂The height of image be a pixel, as shown in figure 11.But in practice, be difficult to keep between the axle aiming at fully, more be difficult to keep aiming at fully as three cameras in the multi-wavelength embodiment of Fig. 8 and 9 demonstrations with between throwing light on going of illuminating line and camera.By in each row of camera a plurality of detector element, for example two to five, be intertwined, when reading noise or time for reading and being subjected to least disadvantage, alignment condition can be relaxed.

Has the spatial filter that one or more rectangle CCD cameras detect as pick-up unit associating variable-width, as the SF in Fig. 8 and 9 ₂And 510 among Fig. 6, each all is installed in the plane with the object plane conjugation, and another advantage of the preferred embodiment will be set forth below.As discussed above, in one embodiment of the invention, detect spatial filter and be removed, line camera (line camera) is used as the combination that detects spatial filter and pick-up unit.But as discussed above, thereby detecting spatial filter, variable-width allows to optimize the signal to noise ratio (S/N ratio) that detection volume is optimized the sample dependence.Following preferred embodiment has kept the advantage of line camera, the i.e. elasticity of speed and variable detection volume.Thereby set the line of enlargement factor height h of a diffraction limit of imaging in the delegation of camera.The width that detects spatial filter is preferably between h≤d≤10h variable.Detecting device on the irradiation post of camera is two-way (binned), before reading, compares with readout time with exposure, and it needs the operation that can ignore the time.

In a preferred embodiment, camera is Princeton InstrumentsNTE/CCD-1340/100-EMD.Read rate is 1MHz under the situation of a small amount of read noise electronics in a preferred embodiment.Pixel format is 1340 * 100, and camera can connect electric wire so that major part row (80%) is removed from survey region, makes camera more effective at 1340 * 20 places.

Except the advantage of reading camera continuously above-mentioned, promptly between obtaining continuously, there be not idle time, another advantage is that its allows to obtain its length only by the rectangular image of range of the sample restriction.This length is determined by the scope of littler camera width and illuminating line.In a preferred embodiment, sample is placed on the bottom, hole of 96 hole microtiter plates, and diameter is 7mm.Irradiated band is 1 μ m * 1mm, and the radiation of sending from irradiation area is imaged onto on the pick-up unit.It is 1mm that optical train is designed to the visual field ²According to the present invention, the image of bottom, hole can produce on the 1 μ m pixel in 1 * 7mm visual field.

Environment control

In a preferred embodiment of the invention living cells is detected.Living cells detects usually to be needed near physiological status so that its normal survival.One of important parameters is a temperature.It is desirable to add a device and can raise and reduce temperature, particularly, the temperature of keeping sample is at 37 ℃.In another embodiment, also need to control relative humidity, and/or CO ₂And O ₂To keep the viability of living cells.In addition, controlled humidity is very important for the small size sample to reduce evaporation.

Describe below and dispose the temperature that can raise, preferably 37 ℃, with three embodiment of the microtiter plate of LCI system compatible.

Imaging system preferably is placed in the cover of lucifuge.In first embodiment, keep sample plane to temperature required by the temperature of keeping in the whole seal closure.Yet,,, limit detection time thereby evaporative cooling will reduce sample volume unless the humidity that raises is kept intentionally at 37 ℃.

Second embodiment has been equipped with heating mantles on microwell plate, it allows microwell plate to move under static cover.Cover on hole top the optical axis of an independent perforate aligming microscope is arranged.This perforate allows to disperse and enters in the movable span, keeps the circulation of carry-over in heating and the confinement plate simultaneously.The space of about 0.5mm allows to move freely microwell plate and reduces evaporation between cover dish that heats and microwell plate.Because the content in observed hole is exposed to ambient condition at most only several seconds by divergence hole, said content can not suffer tangible temperature variation between detection period.

In the 3rd embodiment, a sapphire window very thin, heating is used as the shell of tray bottom.A resistive heater is attached on the dividing plate in hole, and the temperature of keeping window is to required standard.

At another embodiment, three kinds of disclosed methods can differently make up.

The total score orchestration

The video rate structure of imaging system further is configured to inspire dynamic analysis with the transmission of timing reagent in one embodiment, particularly ion channel analysis, and the startup of channel opener enters micropore by transmission solution and finishes.For example, voltage-gated channel can open the after birth depolarization by adding KCl solution.The dependence of channel opener and time of closing subsequently and the respective change of IC often require with enough fast video rate imaging.Yet the proper velocity of imaging system is incoherent, unless pathway reaction can promptly be inspired.

One embodiment of the present of invention provide a kind of total score orchestration.For the operation of on 96 or 384 orifice plates, analyzing, need the additional volume of scope 20-100 μ L.For example the single head divider is an IVEK divider 2000, because relatively be fit to the interpolation of ion channel activity gaonist.Comparable cell can be taken from CAVRO.In general, can distribute a kind of specific compound to give each hole just more satisfactory.An embodiment provides the single head divider on robot motion's device, and this device moves back and forth dispenser head between analysis website, the source plate that comprises specific compound and top cleaning website.The latter is for the cleaning station of fixing top divider with for the cleaning station of disposable top divider.This system relatively inexpensively provides desired function, but its efficient is low, and the suction-preparation of each compound-cleaning cycle needs about 30 seconds.The embodiment that replaces usefulness is integrated into disclosed LCI system such as the such bull divider of Hamilton Microlab MPH-96 and comes by providing.MPH-96 is installed in top divider on the robot motion's device that can carry out above-mentioned suction-preparation-washing cycle, that independently fix by 96 and forms.

In another one preferred embodiment of the present invention, the imaging system that is used in the automatic screening analysis is integrated with such as the such console panel robot of Zymark Twister (plate-handling robots).

The dark field confocal arrangement

Under the situation of lateral resolution less than diffraction limit, the background fluorescence that is caused by suspending liquid can reduce by the application's dark field imaging technique.Figure 12 (a) and 12 (b) show the raypath in traditional dark field.At Figure 12 (a), sample 600 is by the hollow cone illumination from the light 610 of object lens 620.For example, the hollow cone of this light is put an opaque bar 630 by lens 440 places in Figure 10 (a) and is produced.In Figure 12 (b), after the fluorescence that comes out from sample 600 passes the center of object lens 620, be collected.Because illumination is different with the angle of collecting, so have only not only illuminated but also detected plane to be only the plane that comprises sample 600.

Figure 13 (a) and 13 (b) show the raypath in the inverted dark field.At Figure 13 (a), sample 700 usefulness are passed the beam lighting of the light 710 at object lens 720 centers.In Figure 13 (b), only the fluorescence that comes out on every side from object lens 720 outsides is collected into.Collect around from object lens 720 outsides and can for example put an opaque bar 730 and realize by lens 490 in Figure 10 (a).In traditional dark field, the irradiation that inverted dark field is included in an angle with collect in another different angle, also can be detected thereby only sample plane is both illuminated.

Figure 14 describes focal plane in these cases, and the zone bigger than the diffraction limit zone of sample plane of wherein throwing light on is good.Those are identical in the inversion dark field geometric configuration of illumination and the ray collected and Fig. 3.If with the plane of lens back focal plane conjugation with the width that is matched with illuminating bundle on put a diaphragm, then can obtain this dark field architecture.Can recognize that from Figure 14 this architecture has reduced the not fluorescence bump detecting device of coplane.Fluorescence from top shadow region and following object plane does not pass diaphragm.Under the confocal situation of spot scan, reflect effectively from the detected aperture of the fluorescence of these plane exterior domains.Under the online scanning confocal situation, be provided for along the background signal of another point of this line from the not coplane fluorescence along a side position of this line: this is the cause of degenerating aspect signal-background in the line sweep confocal with respect to spot scan.Line sweep confocal inversion dark field architecture has been recovered the pith of the confocal background reflectance attribute of spot scan, the speed advantage that has kept the line sweep architecture simultaneously.

Real time data is analyzed

The present invention can produce the megabytes per second data continuously.In one embodiment, the system integration has quick high strength, mass storage device, real time data can by automatic spool with after analyze.At a preferred embodiment, the operation of data analysis is carried out synchronously with data acquisition basically.Therefore, data are processed before storage.In general, have only the result of analysis to be filed, but its advantage is also to have stored the raw data of selecting simultaneously.

The example of real-time analysis program is provided as follows in conjunction with each analysis bank.In all cases, program is used to the software code of the hardware platform of Optimizing operation research thing.In current preferred embodiment, computing machine is 32 bit processors, as PentiumII.In this case, all data with 32 bit data bags by access.

Generally speaking, the collection of data and analysis comprise many discontinuous steps.At first, fluorescence is converted into one or more digital pictures, and its numerical value is directly proportional with fluorescent radiation intensity on each pixel that incides pick-up unit.In this step because pass the visual field imaging system non-homogeneous response and carried out a correction, wherein the background of disallowable data is known as the file division of flat field.The second, bit figure produces from one of these digital pictures, and wherein all values that satisfy a certain standard replace with 1, and all values that do not satisfy a certain standard are with 0 replacement.At an embodiment, this standard comprises the threshold value of being determined by image self.The 3rd, search for this bitmap to search 1 adjacent value pixel groups.In one embodiment, this group is also tested with respect to minimum and/or full-size standard.The 4th, for qualified group, summation and be recorded in identical image or another image in the value of respective pixel, determine and write down mean value and other statistical property of this summation.Be fit to interpolation and variation various analyses, on this base program with describing below.

Analyze

Many variations of this analytical approach that describes below can be put into practice according to the present invention.In general, distribute with the feature space of one or more fluorescence labeling kinds and/or moment and quantize this analysis.Advantage is that fluorescence is to use the line sweep confocal microscope from being the surperficial observed of plane basically.This cross section is organized by the analysis type that general complicacy according to the related data routine analyzer increases degree.Yet, this organize not strict because this analytical algorithm usually can be used in the more than analysis type.

Binding analysis

First type analysis that the method according to this invention can be operated at an easy rate is a binding analysis.In general, obtain with disclosed line sweep confocal microscope picture system, comprise one or more fluoroscopic images of the sample of target material and tagged ligand at least, quantize the conjugation of fluorescein-labelled part and the target material that is studied.The part that uses comprises: the synthetic analogues of the natural or synthetic peptide of fluorophor combination and protein, sugar, lipid, nucleotide sequence, virus particle, antibiotic plasmid, natural and synthetic toxin, known pharmaceutical agents, organic molecule or neurotransmitter or autofluorescence micromolecule, peptide or protein, the compound, the peptide at random in cDNA library, the protein that synthesize from combinatorial libraries, and peptide (peptidomimetics) (seeing Haugland R.P. fluorescence probe and research compound 6th Ed.Chap.18), but be not limited only to this.The target material comprises: cell extract or acceptor purified reagent, part gate or ion gate channel protein, enzyme, transcription factor, cytoskeletal protein, antibody and the material of deriving from virus, bacterium, antibiotic, no vertebra and vertebrate cells, but be not limited only to this.Exemplary acceptor comprises: as acetylcholine, adrenaline (α and β), muscarine, dopamine, glycocoll, glutamate, compound ammonia, aspartate, gamma-amino tyrosine (GABA), purine (purinergic), histidine, remove to add adrenaline, the P material, neuropeptide tyrosine, enkephalins, neurotensin, cholecystokinin (CCK), endorphin (opioid peptides), melanin (melanocriotin)/ACTH corticotropin, Somat, parathyroid hormone, growth hormone, thyroid-stimulating hormone, thyroxine, the basic element of cell division, (chemokine), insulin, IGF (IGF), stem cell factor, luteinizing hormone-releasing hormone (LRH), promoting sexual gland hormone, angiotensins, Endothelin, neurotensin, interferon, bradykinin, antidiuretic hormone, oxytocins, vasoactive intestinal peptide (VIP), corticoliberim, neurenergen, erythropoietin(EPO), prostaglandin, leukotriene, thromboxane A ₂, calcitonin, T cell, LDL/HDL, epidermal growth factor (EGF), estrogen and galactonic acid (galainan), but be not limited only to these.

Combination (bingding) based on silica bead

Figure 15 (a)-15 (f) has described the step of the embodiment of the receptor binding assay that can operate according to the present invention.In Figure 15 (a), be added in the hole 220 that comprises liquid 230 by cell or tissue film 210 preparation, that comprise target acceptor 212.In Figure 15 (b), fluorescein-labelled part 214 is added in the hole 220; These part binding film acceptors 212.In Figure 15 (c), silica bead 224 is added in the hole 220.In addition, 15 (b) can put upside down to the order of 15 (c), and in a preferred embodiment, the silica bead of film bag quilt is to be separated to prepare before adding in the hand-hole.The diameter range of silica bead 224 approximately is 1-20 μ m, is covered with for example wheat germ agglutinin of layer of substance, and film 220 can be adhered on it, or has a surface to allow directly covalently or non-covalently combination of film.

Abovementioned steps is except with the fluorescein-labelled replacement radioactive label, and all the corresponding steps of the prior art SSA that describes with Fig. 1 (a)-1 (f) is the same.Therefore but in the present invention, silica bead 224 is non-luminous, and they have certain density, and they can be deposited to the bottom in hole, thereby or is magnetized and when using external magnet they is moved at the bottom of the hole.In Figure 15 (d), fluorescein-labelledly be used the line sweep confocal microscope imaging that for example is described as element 240.In Figure 15 (e), a testing compound 218 is added in the hole.As the analysis of prior art, the purpose of this analysis is to judge the degree of testing compound from membrane receptor 212 displacements fluorescein-labelled 214.In Figure 15 (f), still be combined in fluorescein-labelled on the film 210 by imaging.By comparing two fluoroscopic images, can determine the activity of testing compound.

In another embodiment of the analysis that Figure 15 (a)-(f) describes, the image-forming step of describing at Figure 15 (d) can omit, the activity of testing compound can be by image and the control wells image that Figure 15 (f) is obtained, or recently definite mutually with the known expection image that adds the affinity of fluorescein-labelled amount of ligand in the hand-hole and known they and acceptor and obtain.

In a special embodiment of the analysis that Figure 15 (a)-(f) describes, but acceptor is the antibody of recognition ligand, and react to fluorescein-labelled together the adding with the sample that comprises the unmarked part of non-quantitative.As existing radioimmunoassay technique, the purpose of this analysis is to determine the concentration of unmarked part in the sample by the degree of measuring the fluorescein-labelled part 214 of being replaced from antibody receptor.

Surface combination

Figure 16 (a)-16 (f) has described the step according to second embodiment of receptor binding assays of the present invention.In Figure 16 (a), by cell or tissue preparation, the film 250 that comprises target receptor 25 2 is added in the hole 260 that comprises liquid 270.Hole bottom 262 scribbles layer of substance for example can make film adhere to wheat germ agglutinin on it.In Figure 16 (b), shown that film 250 is combined on this layer material.In Figure 16 (c), fluorescein-labelled part 254 is added in the hole 260 and in conjunction with this membrane receptor 252.In addition, the order of 16 (b) and 16 (c) can be put upside down.

In Figure 16 (d), fluorescein-labeled fluorescence is used the line sweep confocal microscope imaging that for example is described as element 280.In Figure 16 (e), a testing compound 258 is added in the hole 260.In Figure 16 (f), still be combined in fluorescein-labelled on the film 250 by imaging, and with first image relatively, determine the activity of testing compound 258.

In another embodiment of the analysis that Figure 16 (a)-(f) describes, image-forming step at Figure 16 (d) can omit, the activity of testing compound can be by image and the control wells image that Figure 16 (f) step is obtained, or recently definite mutually with the known expection image that adds the affinity of fluorescein-labelled amount of ligand in the hand-hole and known they and acceptor and obtain.

Combination based on cell

In another embodiment, can analyze part-target material combination at an easy rate by collecting the cell of expressing the target material.In general, carry out the lot of advantages that is combined with the scanning compound based on cell.Particularly competition and compensatory these two kinds when influencing the bioactive programmed cell of compound and all existing, the activity of measurement Research thing.In cell analysis, be placed in the tissue culture hole or on the microscopical microslide, cell can be survived and be without prejudice from the cell of clone or tissue preparation, perhaps can be by reagent infiltration such as digoxin one class, or available formaldehyde fixed.The reagent of the no fluorescein that one or more fluorescein-labeled parts and any analysis are required is added into cell together; Fluorescein-labelled part is in conjunction with one or more cell internalizing compounds.Then testing compound is added cell.In addition, the addition sequence of fluorescein part and compound can be put upside down.Use the line sweep confocal microscope imaging of for example element 240 descriptions fluorescein-labelled.The purpose of this analysis is to measure the concentration of the fluorescein-labelled part that testing compound replaces from acceptor.Be imaged on to be with or without and still be combined in fluorescein-labelled on the cell under the situation that testing compound exists,, can determine the activity of testing compound by these two fluoroscopic images relatively.

In another embodiment based on the receptor binding assays of cell, can omit the image-forming step that does not have under the compound situation, image and control wells image that the activity of testing compound can obtain by will have compound the time, or recently determine mutually with the known expection image that adds the affinity of fluorescein-labelled amount of ligand in the hand-hole and known they and acceptor and obtain.

The advantage of line sweep confocal microscope in binding analysis

At first embodiment, carry out part-target material combination with an excitation wavelength and an emission wavelength.The data instance that Figure 24 provides speed of the present invention and susceptibility.Its labor of performance that below description is related to prior art, wherein part with radioactive label so that detect.The receptor-ligand analysis of prior art comprises the SSA form, and with physical method with combination and not binding partner separately and be attached to the form of the amount of ligand on the acceptor with adding liquid scintillator mensuration.

At first, the present invention can be used for the low capacity hole, and for example 1 μ L is using radioactive mark ligand's receptor-ligand binding analysis, each radioactive label, for example ³H only once will decay, and each decay produces maximum 90 photons, and the per second attenuation rate is less than 10 ^-8A single fluorescein molecule can produce 10 altogether ⁴～10 ⁷Individual photon, its per second will launch 10 ³To 10 ⁶Between photon.Therefore, with respect to ³The fluorescein-labeled counting rate of H approximately is 10 ¹¹Therefore, in the present invention, every hole only needs mark, film and silica bead seldom.For example, when the every hole 10 of tritium SSA needs ⁷During individual silica bead, the every hole of the present invention only need be less than 10 ³Individual silica bead.As a result, the present invention only just can carry out in μ L-capacity hole and very short time.In addition, be difficult to change imaging time at SSA, because radioactive label is decayed with fixed rate.On the contrary, can increase the emissivity of fluorescein-labeled excitation rate, thereby reduce required imaging time with the increase photon.Yet excitation rate can not unrestrictedly increase.In fact, in this application, have the saturation limit of so-called fluorescence excitation rate, Here it is, and line sweep is better than the spot scan part.The second, thing of the present invention takes time and spends and handles radiomaterial.The 3rd, because the present invention can operate in the low capacity hole, expending than SSA of compound and reagent significantly reduces, and further reduced expense.At last, the present invention does not need to be coated with the silica bead or the bottom of luminous agent, and expense further reduces.

The present invention uses the line sweep confocal microscope to come the fluorescence of sample in the imaging hole.Microscopical confocal feature allows optical segmentation, promptly detects fluorescence from the residing plane of sample, reduces and detect fluorescence from large quantities of solution.This has saved will remove the not rinsing step of combined with fluorescent tagged ligand.This step still needs in other any receptor-ligand binding analysis except not needing in SSA, comprises the RIA of the pearl of need not glimmering.This microscopical confocal feature has also been got rid of the interference problem of the testing compound autofluorescence that may occur.Line sweep characteristic permission sample can be to scan fast velocity imaging than conventional point when not losing noticeable background reflectance.The increase of speed depends on fluorophor intensity, lateral resolution, the visual field, hardware parameter and comprises object lens NA, detection sensitivity and camera read rate.Theoretically, the increase of speed can reach the number of every capable pixel, and this is 1000 in the preferred embodiments of the present invention.In fact this increase approximately is 100X.

In order to quantize these advantages, a sample example will be described below.Analysis is based on cell, and wherein the location of fluorescence can be accurate to 1 μ m.Therefore the imaging of 1mm diameter sample area will be by～10 ³Pixel～10 ³Line constitutes.The fluorescence signal that is studied object may be from the source of the part or the cell internal fixation of cell surface, for example nuclear receptor.In another case, the local concentration of fluorophor is an important parameters.For the every cellular expression of a genetic engineering (engineered) clone～10 ⁵Individual acceptor, cell mean concentration (cell-averaged) are-1 μ M.Several thousand acceptors in being positioned to examine cause considerable local concentration.Meeting 1 required μ m lateral resolution, every pixel is nearly～and 2 * 10 ³Fluorophor.The fluorophor of supposing cell background self is less than mark fluorescent, but at an order of magnitude, required signal to noise ratio (S/N ratio) minimum is 10.Take into account the sudden strain of a muscle grain noise of signal and the read noise of background and high frequency solid-state detector, the number of detected photon almost need be up to 10 ³The present invention use about 0.7NA object lens, stop the collection of light filter and solid state camera and verification and measurement ratio be～1%.Need every pixel about 10 ⁵Photon, or per molecule～10 ²Photon is launched.Can be more preferably part and obtain imaging in the time of second less than one second.If obtain pixel in serial mode, then the pixel hold-up time must need the per second per molecule greater than 10 less than 1 μ s ⁸The photo emissions rate.This has surpassed fluorophor typical 10 ⁶Maximum saturation value.The flow 10 that importantly, need reach capacity ⁵～10 ⁶W/cm ², equally also enough drive the fluorophor that non-linear photon induces and discolor.As a result, can not use pick-up unit the most efficiently under the data rate that in series scanning, needs.On the contrary, if 10 ³Pixel is shone synchronously, and then the emissivity of each fluorophor only needs 10 ⁵The confocal background fluorescence of spot scan disturbs the increase of (rejection) can not overcome the shortcoming of sweep velocity sharp fall.

The data display that Figure 24 exemplifies the system that set forth enough sensitivity is arranged, to quantize tens thousand of fluorophors of every pearl, in less than one second time, clearly analyze hundreds of independent silica beads simultaneously.Can obtain equal data at cell-bassed in conjunction with test, will illustrate below.

Data analysis

DAP with based on cell also be based on silica bead or both all have simultaneously closely relatedly, will describe below.Data can be used the following procedure analysis, and the simplest is the analysis of threshold algorithm.The purpose of this program is to judge the amount of the fluorescence labeling kind of locating in the mode of continuous or point-like, surpassing minimum fluorescence intensity, but surpasses maximum fluorescence intensity.In one embodiment, this analysis is used to the activity of analysis of compounds.

This algorithm steps is as follows:

1. obtain the digitized image of this mark kind.

Line by line opening document and

I. from image, deduct the camera off-set value.

Ii. the row of each in the image multiply by the inverse of corresponding row in the flat field image document.

3. randomly, this image is made histogram to determine the background standard.

4. set up and comprise minimum value and randomly peaked choice criteria.Determining this value by statistical study background histogram peak width or use predetermined value, for example is the fixedly multiple of average background standard, or the fixed number of the counting on the average background standard.

5. each pixel in the image and choice criteria are compared.If each pixel in the image is conformance with standard all, then add this and be worth the operation sum.Report standard compliant sum of all pixels and mean intensity.

This program be used in easily handle with Figure 24 in similar data, wherein single silica bead quilt is very clearly distinguished from background or artefact, because silica bead in heaps or cell are less.Such program is fit to the analysis type that film is attached to the bottom, hole equally.

Second program that can be applicable to analyze binding data is the local analytical algorithm, and it needs other routine analyzer.The same with the threshold value program, the purpose of this program is to judge the amount of the fluorescence labeling kind of locating in the mode of continuous or point-like, and in one embodiment, this analysis is used to the activity of analysis of compounds.

This algorithm steps is as follows:

1. obtain the digitized image of this mark kind.

Line by line opening document and

I. from image, deduct the camera off-set value.

Ii. the row of each in the image multiply by the inverse of corresponding row in the flat field image document.

3. randomly, this image is made histogram and summation pixel value.

4. set up and comprise minimum value and randomly peaked choice criteria.Determine this value by statistical study background histogram peak width or use predetermined value, for example be the fixedly multiple of average background standard, or be the fixed number of the counting on the average background standard.

5. each pixel in the image and choice criteria are compared.All standard compliant pixels all are assigned 1, and other is assigned 0, therefore realize 16 to 1 compression.

6. by will the whole values in binary mask being that 1 adjacent pixels is arranged to 0, come " removing " image border with engagement edge element.

7. the search for items bitmap defines the pixel of continuous group for value 1, by:

1. with line by line mode searching image pixel with discovery value 1.

2. judge the pixel of all values 1 and the continuous pixels of in i, discerning.

3. randomly use minimum and full-size light filter on article, this size is scheduled.

4., carried out for the 8th step, otherwise to change 1 value pixel in all article be 0 and continue to search for next article if article are qualified.

5. if arrive last bitmap, then operating procedure 9.

8. to each article by the light filter standard:

1. randomly create a new rectangular bitmap that contains extended boundary, the extended boundary of this bitmap comprises the target that adds n extra 0 pixel from each direction at article edge.N be below will move, and the number of the expansion step reserved in advance.

2. if step 8.i is performed, expand article n time by using expansion step, the 0 value pixel that wherein contacts 1 value pixel is set to 1.

3. if the 8.i step is not carried out, then at the expansion bitmap or in each set of 1 value pixel of initial bitmap, summation with on average from the respective pixel of this image so that under mask compute average pixel intensity.

4. conversion all pixels in initial bitmap images become 0, and turn back to step 7, so that search for more article.

9. after all article all are counted, to all article in the image, calculate any part of total intensity of the kind of the mean intensity of fluorescence labeling kind of each article and location, and it and the statistical information such as standard deviation are together reported.

Difference in this program between the shared operation of all following algorithms is the establishment of the scale-of-two mask of step 4-6.Can comprise minimum and maximal value, size and dimension for the article choice criteria of this mask.For example, at an embodiment, be used for comprising the round light filter of step 7iii based on the routine analyzer of pearl journey (based on silica bead) analysis.

At second embodiment, the emission of two or more fluorescence labeling kinds that synchronous detection is excited by one or more illumination wavelengths.As using in the binding analysis, the first fluorescence labeling kind is used to discern the article of the second fluorescence labeling kind combination.Provide two two color bases in the binding analysis of cell at Figure 21 and 23.Program example that can be used to analyze this image is altogether-local routine analyzer (Co-localization Analysis), and its design is used for judging the first fluorescence labeling kind with respect to second fluorescence labeling kind location.At an embodiment, this analysis is used to the activity of analysis of compounds, for example depends on the compound that is studied of Subcellular Localization in activity.

This algorithm steps is as follows:

1. obtain the digital picture of first and second kinds of marks respectively.

Line by line opening document and

I. from each image, deduct the camera off-set value.

Ii. the row of each in each image multiply by the inverse of corresponding row in the flat field image document.

3. randomly first image is made histogram to judge the image intensity of background standard and summation second kind.

4. set up and comprise minimum value and randomly peaked choice criteria.Determining this value by statistical study background histogram peak width or use predetermined value, for example is the fixedly multiple of average background standard, is the fixed number of the counting on the average background standard.

5. each pixel in the first familygram picture and choice criteria are compared.All standard compliant pixels all are assigned 1, and other is assigned 0, therefore realize 16 to 1 compression.

6. by will the whole values in binary mask being that 1 adjacent pixels is arranged to 0, come " removing " image border with engagement edge element.

7. search for items bitmap is defined as the pixel groups of continuous value 1, by:

1. mode searching image line by line is with the pixel of discovery value 1.

2. judge the pixel of all values 1 and the continuous pixels of in i, discerning.

3. the optional minimum and full-size light filter of use on article, this size is scheduled.

4., carried out for the 8th step, otherwise to change 1 value pixel in all article be 0 and continue to search for next article if article are qualified.

5. if arrive last bitmap, then operating procedure 9.

8. to each article by the light filter standard:

1. randomly create a new rectangular bitmap that contains extended boundary, the extended boundary of this bitmap comprises the article that add n extra 0 pixel from each direction at article edge.N be below will move, and the number of the expansion step reserved in advance.

2. if step 8.i is performed, expand article n time by using expansion step, the 0 value pixel that wherein contacts 1 value pixel is set to 1.

3. if the 8.i step is not carried out, then at the expansion bitmap or in each set of 1 value pixel of initial bitmap, summation with on average from the respective pixel of the image of second kind so that under mask compute average pixel intensity.

4. conversion all pixels in initial bitmap images become 0, and turn back to step 7 so that search for more article.

9. after all article all are counted, to all article in the image, calculate each article the second fluorescently-labeled kind mean intensity and with any part of the total intensity of second kind of the first kind co, and it and the statistical information such as standard deviation together reported.

This more the advantage of detailed procedure be, though these article it be that cell or silica bead can both be identified independently.Example as Figure 21: not every cell all responds.For example, the independent identification of cell will make the degree of reacting in the ratio of reaction and reacting cells not and those reactors together make form.This algorithm, other complicacy that let it be can be carried out on Pentium Ц platform with one second time inner analysis 1,000,000 pixel.

Transposition is analyzed

Can carry out other analysis type easily according to second embodiment, promptly the emission of the two or more fluorescence labeling kinds that excited by one or more illumination wavelengths is by the transposition analysis of synchronous detection.In these were analyzed, the transposition that is studied material was one or more kinds, and it can be protein, lipid or other molecular complex or subcellular structure such as vesica, from the zone of the sharp outline of a cell to another.This including but not limited to: cynapse (synaptin) (vesica memebrane protein), transcription factor (NK-kB, NFAT, AP-1), hormone receptor, LDL/HDL acceptor, TXi Baoshouti and PTH acceptor.

The transposition analysis of prototype is the special circumstances that common-localization is measured.For example, first and second kinds altogether-localization by with respect to second kind of first kind altogether-that part of localization quantizes, perhaps second kind and first kind and be present in the cell other places altogether-the ratio quantification of localization.Provide below than the extensive diagnostic program that is more suitable for handling the transposition view data.

This mark is positioned nucleus, and this mark is the fluorescence special to DNA, for example Hoechst33342.Other dyestuff to nucleic acid specificity is that this area is known (as Haugland, R.P. fluorescence probe and research chemistry, sixth version, the 8th chapter) altogether.Second kind is transcription factor, and it is the object of analyzing to nuclear migration from tenuigenin.This albumen can be used the several different methods mark, comprises the expression of merging with GFP, and contact is to the sample of the special fluorescent-labeled antibody of transcription factor protein.

Following transposition DAP (translocation Data Analysis) can be used to judge first kind of fluorescently-labeled amount to distribute with second kind of relevant or incoherent mode of fluorescence labeling.At an embodiment, this analysis is used to the activity of analysis of compounds.

1 obtains the digital picture of first and second kinds of marks respectively.

2 line by line opening document and

I. from each image, deduct the camera off-set value.

Ii. the row of each in each image multiply by the inverse of corresponding row in the flat field image document.

3 randomly look like first familygram to make histogram to judge the image intensity of background standard and summation second kind.

4 foundation comprise minimum value and any peaked choice criteria.Determining this value by statistical study background histogram peak width or use predetermined value, for example is the fixedly multiple of average background standard, is the fixed number of the counting on the average background standard.

5 with each pixel in the first familygram picture and choice criteria comparison.All standard compliant pixels all are assigned 1, and other is assigned 0, therefore realize 16 to 1 compression.

6. by will the whole values in binary mask being that 1 adjacent pixels is arranged to 0, remove the image border with engagement edge element.

7 search for items bitmaps are defined as the pixel groups of continuous value 1, by:

1 line by line mode searching image is with the pixel of discovery value 1.

2 judge the pixel of all values 1 and the continuous pixels of discerning in i.

The 3 optional minimum and full-size light filters of use on article, this size is scheduled.

If 4 article are qualified, carried out for the 8th step, otherwise to change 1 value pixel in all article be 0 and continue to search for next article.

If 5 arrive last bitmap, then operating procedure 9.

8. to each article by the light filter standard:

1 creates a new rectangular bitmap that contains extended boundary, and the extended boundary of this bitmap comprises the article that add n extra 0 pixel from each direction at article edge.N be below will move, and the number of the expansion step reserved in advance.

2 expand article n time by using expansion step, and the 0 value pixel that wherein contacts 1 value pixel is set to 1.

3 relatively expand bitmap and initial actual size bitmap.Be arranged on all pixels of expanding in the way promptly in initial bitmap 1 value of corresponding region be 0.Produce an annular mask like this, guarantee to have only article to be hunted down when the bitmap edge increases during expanding.

4 create another bitmap from original goods, by the 1 value pixel that contact 0 value pixel is set be 0 erosion it m time.M is equal to n, determines in front.

5 in annular or in each set of 1 value pixel of the bitmap that corrodes, summation with on average from the respective pixel of the image of second kind, with corrode or annular mask under compute average pixel intensity.

6 calculate that each article corrodes to the ratio of annular bitmap and be stored in the table.

7 conversions all article pixels in initial bitmap images become 0, and turn back to step 7 so that search for more article.

9 after all article all are counted, the mean intensity ratio of all article in the computed image, and it and statistical information such as standard deviation together reported.

The new feature that this program is different from said procedure is that one is the annular expansion of original mask in the establishment of the 8th step two sub-masks, and one is the pattern of the erosion of original mask.In this example, back a kind of be used to quantize kind 2 and kind 1, the common-localization of transcription factor and nucleus (being actually DNA).Previous mask is used to quantize not have the kind 2 of common-localization.In present example, the ratio of these two kinds of quantifications is to form on the cell base one by one, and the result is made a table.

The method according to this invention, the collection of data and analysis almost can be finished in 1 time in second.For relatively, quote the example of two prior aries here.In (J.Bio1.Chem, 273,28897-28905 (1998)) such as Ding, carried out one two look transposition and analyzed.Advantage of the present invention comprises: 1) every image channel almost the rapid image of 50X obtain 2) synchronous two color images obtain 3) the about hypersensibility of 10X, allow low standards for dyeing.4) confocal detection allows to save rinsing step, 5) the about 30 seconds focal time of contrast, the present invention only needs 0.1 second focal time.6) data-analysis time of contrast 3-6s/ frame, it only needs 0.2s/ frame, 7) consecutive image obtains.Second example of prior art is (Cytometry, 33,376-382, (1998)) such as Deptala.The invention provides: 1) high spatial resolution is about 4X, 2) the high pixel that is approximately 16X obtains rate, 3) the rapid data analysis, 4) exercisable autofocus system and 5 on the microtiter plate) data-analysis time of contrast 3-6s/ frame, it only needs the 0.2s/ frame.

Endocytosis, exocytosis and acceptor chelating

In general, endocytosis, exocytosis, acceptor chelating and recycle are the special appendages that can analyze according to first or second embodiment and above-mentioned associated picture analytical plan.Fluorescence labeling can be finished according to various known methods.For example, by disclosed first-class tests that comprises acceptor and ligand-labeled such as Tarasova.The every image channel of this image analysis system is 50X soon, but two images of synchronization gain.In addition, this analysis scheme, for example common-localization algorithm can be used to handle in real time the chelating view data.There is not such example in the prior art.

Many other known in the state of the art analyzed and needed similar image and analysis ability.For example, phagocytosis and relevant cellular activity, (J.Immunology for example, (1983) 130,1910; J, Leukocyte Biol. (1988) 43, and 304); (for example Neuron 14,983 (1995) to comprise receptor-mediated and non-receptor-mediated endocytosis and exocytosis analysis in addition; J.Physiol.460,287 (1993) and Science 255,200 (1992)), comprise that low-density lipoprotein compound (Low-Density Lipoprotein Complexes) (sees J.Cell Biol.121,1257 (1993) and the transfection transmission (seeing Cell 49,423 (1994)) of vertebrate cells; The endocytosis of fluorescently-labeled epidermal growth factor and the imaging of transverse movement (are seen Proc.Natl.Acad.Sci.USA75,2135 (1975); J Cell Biol.109,2105 (1989)); There are the picked-up and the inter-process of fluorescence glucosan encytosis monitoring exocytosis material (to see J.Biol.Chem.269,12918 (1994)), imaging (seeing Nature 314,357 (1985)) with the synaptic versicle recycle of the endocytosis mediation of hydrophilic dye in activating the neuron process.In addition, the tPA (seeing Mol.Biol.Cell 9:2463 (1998)) of genetic engineering that expression of cell lines green fluorescent protein (GFP) and the albumen (seeing J.Cell Sci.110,1453 (1997)) that is arranged in exocytosis and excretion vesicles merge or the nerve growth awl that is positioned to break up allows the monitoring exocytosis.Numerous fluorescence labelings can be used for such analysis (seeing Haugland, R.P. fluorescence probe and the chemical handbook of research, sixth version, the 17th chapter).

Ion channel

The 3rd embodiment of the present invention, one of pattern of describing at Fig. 9 can be used to be imaged on the time dependent reaction of the next or a plurality of fluorescence labeling kinds of the speed of about 30 frames of per second.This allows to catch the transient phenomenon such as opening or close ion channel.The ion channel of example comprises: K ⁺-gate voltage, Na ⁺-gate voltage, Ca ⁺⁺-gate voltage, Cl ^-, Na ⁺/ K ⁺ATP enzyme and P glycoprotein.

Below dynamic imaging data analysis (Kinetic Imaging Data Analysis) algorithm definition and follow the tracks of the individual cells of a frame one frame, cell that can the enough numbers of synchronous dynamics analysis is to obtain the satisfied data that statistical significance is arranged.This algorithm steps is as follows:

1 obtains one (only indicator), two (label and indicator or two indicator) or a plurality of digital picture function as the time.

2 line by line opening document and

I. from each image, deduct each camera off-set value.

Ii. the row of each in each image multiply by the inverse of corresponding row in each flat field image document.From each image, deduct each camera off-set value.

3 randomly look like first familygram to make histogram to judge the background standard.

4 foundation comprise minimum value and any peaked choice criteria.Determining this value by statistical study background histogram peak width or use predetermined value, for example is the fixedly multiple of average background standard, is the fixed number of the counting on the average background standard.

5 with each pixel in the first familygram picture and choice criteria comparison.All standard compliant pixels all are assigned 1, and other is assigned 0, therefore realize 16 to 1 compression.

6 by will the whole values in the binary mask with engagement edge element being that 1 adjacent pixels is arranged to 0, comes " removing " image border.

7 search for items bitmaps are defined as continuous class value 1 pixel, by:

I. mode searching image line by line is with the pixel of discovery value 1.

Ii. judge the pixel of all values 1 and the continuous pixels of in i, discerning.

Iii. the optional minimum and full-size light filter of use on article, this size is scheduled.

If iv. article are qualified, carried out for the 8th step, otherwise to change 1 value pixel in all article be 0 and continue to search for next article.

If v. arrive last bitmap, then operating procedure 9.

8. to each article: the respective pixel of each image in the time series is average by the light filter standard.If use single indicator, write down this intensity.If use radiometric indicator, the value that each image in the time series is divided an image with the value of another image, and record result.

9 after all article are all analyzed, the analysis result of each article of reporting step 8.Report the dynamic parameter that comprises flush time, die-away time and amplitude of each article, as from a collection of dynamic analysis with from all article of cover statistical information of deriving of set time.

Figure 19 and 20 provides and has used two examples of the present invention with imaging and the analysis transient state action relevant with ion channel.These are analyzed and use Ca ⁺⁺Quick dyestuff, Fluo-3 come Ca in the indicator cells ⁺⁺Concentration change.In first test, variation is because the Ca that the activation of acetylcholinergic receptor inspires ⁺⁺Secondary signal causes.Second test, this variation is by valtage-gated Ca ⁺⁺The activation of passage causes.

Ion channel is the hot fields of recent researches.The present invention with respect to the advantage of prior art below relatively after can become clearly.

In screening compound is used, quote the prior art standard of background technology part, be at U.S. Patent number 5,335, disclosed in 215.This device is used for detecting Ca in the cell at first ⁺⁺Induction change, comprise a divider that is used to inspire the transient state action.It is as follows that the present invention is better than the main point of prior art: 1) imaging and analyzing is allowed by relatively judging the reaction of individual cells with the average response level in this hole.2) increase of susceptibility only needs to add a spot of reagent and low intensity of illumination, and required sample size also reduces.3) obtain image with video rate, can compare with the maximum rate of 1 of per second.

In research is used, at biological confocal microscope handbook (Handbook of BiologicalConfocal Microscopy) J.B.Pawley, ed., Plenum publishing house, New York, 1995, disclosed Tsien system and co-worker are as standard among the pp.459-478.It has shown to have to surpass the ability of speed imaging of the present invention.Yet this can not be used in the sample of present research.Prior art need be a comparable signal to noise ratio (S/N ratio), every pixel 10 ²-10 ³Bigger fluorophor is to reach speed of the present invention.In addition, the present invention can provide under the bigger dynamic range situation of 4-16X and obtain image at 12-or 16-position resolution.

Second example of research system be at Sun etc., J.Physiology, and 509,67-80 is disclosed in 1998..According to Sun, use traditional spot scan confocal microscope, can be in speed up to the 650Hz/600 pixel line, with 5 microseconds/pixel integration time generation data.Can only produce the one dimension image.Can monitor the transient phenomenon that is positioned at the article on the sweep trace.In addition, this speed only just can reach in 1 μ s pixel integration time, needs 10 ²-10 ³Big fluorescence concentration is to reach the picture quality that can compare with the present invention.

The ability that the interior ion concentration of imaging of the present invention and analysis of cells is replied the outside stimulus variation has multiple application (for example to see J.CeIl Biol.137 (3), 633-648 (1997) in screening compound and general biological study application; J.Biol.Chem.271 (9), 4999-5006 (1996); Science280,69-76 (1998); Biochem, J.324,645-651 (1997)).Many fluorescence indicators are used in the test to the specific ion sensitivity (sees Haugland, R.P. fluorescence probe and the chemical handbook of research, sixth version, the 18th, 22 and 24 chapters).These indicator are allowed Mg ²⁺, Zn ²⁺, Ca ²⁺, Na ⁺, Fe ²⁺, Hg ²⁺, pb ²⁺, Cd ²⁺, Ni ²⁺, Co ²⁺, Al ³⁺, Ga ²⁺, Eu ³⁺, Tb ³⁺, Sm ³⁺And Dy ³⁺Concentration determination.In addition, even under the situation that the univalent cation that other physiological concentration is arranged exists, also can analyze Na ⁺And K ⁺(seeing J.Biol.Chem.264,19449 (1989)) comprises and analyzes for example Na in haemocyte, brain cell and the muscle cell of various cells ⁺Level or Na ⁺Flow and (to see J.Biol.Chem.268,18640 (1993); J.Neurosi.14,2464 (1994); Am J.Physiol.267, H568 (1994)) K and in spermatoblast, nerve endings synaptosome and the lymphocyte ⁺Variation.In addition, the present invention can be used in analysis Cl in vesica, liposome and living cells ^-Concentration (seeing Am.J.Physio.259, c375 (1990)).

In addition, the present invention also can be used to the variation of film potential on analysis of cells and the organelle.The variation of imaging film current potential promptly is that the pass of generation, contraction of muscle, cell signal and ion gate hole path of viability, nerve impulse of analysis of cells and organelle is in conjunction with (seeing Biophys J.67,208 (1994); Neuron 13,1187 (1994); J Membrane Biol.130,1 (1992)).Fluorescence labeling can be used to excitable cell such as quick (microsecond) film potential variation of neuron, cardiac muscle cell and the generation of undamaged brain cell are replied (seeing Haugland, R.P. fluorescence probe and the chemical handbook of research, sixth version, the 25th chapter).The fluorescence probe of replying quick membrane potential variation only shows that the fluorophor that is generally the about 2-10% of every 100mV changes.The cell plasma film has approximately-membrane potential of 70mv, and for example mitochondrial membrane potential of some organelles can keep-150mV.Therefore, comprise that this fast-changing analysis needs high sensitivity, the fast data acquisition capacity, this is very usual for various embodiment of the present invention.

The mensuration that can transmit based on fluorescence resonance

The present invention can be used at an easy rate carrying out and comprise that fluorescence resonance can transmit the analysis of (FRET).When a fluorophor, donor is transferred to another fluorophor with absorbing a photon and the energy on-radiation that will absorb, and FRET then takes place acceptor.Then, this receptor is launched this energy with its characteristic wavelength.Donor and acceptor molecule must be very approaching, less than 10nm, are beneficial to the generation that effective energy shifts.(see Methods Enzymol.211,353-388 (1992); Methods Enzymol.246,300-334 (1995)).This required vicinity can be used to found for D-A between the analysis of little gap sensitivity.FRET typically needs single excitation wavelength and two emission wavelengths, and an analysis comprises the ratio of donor and acceptor emissive porwer.The FRET D-A to can be used to found not only based on silica bead and also based on the analysis of cell.Green fluorescent protein (GFP) mutant of the fluorescence that several demonstrations strengthen and the emission wavelength of change can be by merging GFP FRET donor and an albumen, GFP FRET acceptor merges with identical albumen or another albumen of the same cell inner expression that coexists merges, and with the analysis pairing of FRET based on cell.Such FRET changes in the analyzing molecules can be used to, as Ca ²⁺Ca ²⁺Combination of-regucalin or intermolecular interaction turn usefulness into as receptor dimerization.Above disclosed dynamic imaging algorithm can preferentially use.

Transient transfection

In the mensuration based on image, the most significant advantage is not only to have an opportunity to observe the incident of the preciousness that can lose generally speaking, and the first order reaction that can standardize on the basis of article one by one becomes second order reaction.These two features have in the analysis of clone of target material of transient transfection all very important in use.The generation of the gene expression after the transfection and the protein of secondary usually is invalidly and instantaneous (to see Bio Techniques 24; 478-482 (1998)).The method of the monitoring transfection efficiency that can use at an easy rate according to the present invention is known in the art.For example, being studied gene can be with the transfection of green fluorescent protein (GFP) gene, and these two albumen will be expressed as expressing fusion protein or the entity that separates like this.The present invention can be used to detect the amount of the indicator that is present in a wavelength place and locates the reaction relevant with the target material at another.Can be used to the to standardize reaction to existing target amount of back of the signal of front.This allows that the present invention carries out the analysis to the target material, to such an extent as to this target material is because its unstable can not and can not detect the only transfection efficiency of a few percent with the screening technique of current use.Above disclosed dynamic imaging algorithm can be used to analyze such data, only need a picture frame here.Can monitor the virus infections of cell, or the expression directly by virus protein, or indirectly by obtaining new phenotype, even only have only cell seldom infected.At last, the invention provides and be used to detect precious incident, for example obtain new phenotype, this new phenotype is owing to whole cell mass is caused individual cells or the special cDNA of a group cell transfecting to obtain by different cDNAs library transfection results.

Enzyme is analyzed

The present invention can be used to carry out the analysis of general enzymatic activity.Exemplary desmoenzyme comprises: carbonic anhydrase, guanosine be in conjunction with albumen (G albumen), adenyl cyclase, regucalin, PI, PIP and PIP2 kinases, tyrosine protein kinase, phosphoprotein phosphatase, beta-lactamase, β-nougat, dihyrofolate reductase, PDE, lime feldspar (caspase), proteosome proteinase, oxides of nitrogen synthase, thymidine kinases, deaminase, glutathione s transferase, lipoxygenase and phosphatidase, but be not limited only to this.

Figure 17 (a)-17 (b) has described the step of first embodiment that analyzes according to enzyme of the present invention.In Figure 17 (a), the silica bead 310 that is coated with the fluorescence labeling peptide 312 of dose known amounts is added in the hole 320 of containing liquid 330.The density that silica bead 310 has makes them deposit to the bottom in hole.In Figure 17 (b), testing compound 314 is added in the hole.In Figure 17 (c), enzyme 316 is added in the hole.Can not add in hole before not having testing compound peptide or the enzyme, the order of the step that Figure 17 (a), 17 (b) and Figure 17 (c) describe can be exchanged.If without limits, then enzyme 316 will divide peptide 312, and with the fluorescence labeling mixing in liquid.On the other hand, if testing compound 314 restriction enzymes 316 are typically the avtive spot that has sealed enzyme, enzyme 316 can not divide fluorescence labeling.In Figure 17 (d), still be coated on the fluorescence labeling on the silica bead, for example, using as 340 elements of describing among the figure is the imaging of line sweep confocal microscope.From this image, can judge the activity of testing compound.

In another embodiment that the enzyme that Figure 17 (a)-17 (d) describes is analyzed, the activity of testing compound can relatively be determined by the image that will obtain in Figure 17 (d) and image or control group image that the fluorescently-labeled fluorescence of imaging in Figure 17 (a) or 17 (b) obtains.

Figure 18 (a)-18 (d) has described the step of second embodiment that analyzes according to enzyme of the present invention.In Figure 18 (a), the fluorescence labeling peptide 352 that is coated with dose known amounts is coated in the hole 360.In Figure 18 (b), testing compound 354 is added in the hole.In Figure 18 (c), enzyme 356 is added in the hole.In Figure 18 (d), still be coated on the fluorescence labeling of bottom, hole, for example, using as 380 elements of describing among the figure is the imaging of line sweep confocal microscope, to judge the activity of testing compound 354.

In another embodiment that the enzyme that Figure 18 (a)-18 (d) describes is analyzed, the activity of testing compound can relatively be determined by the image that will obtain in Figure 18 (d) and image or control group image that the fluorescently-labeled fluorescence of imaging in Figure 18 (a) or 18 (b) obtains.

The example of another analysis that can carry out according to the present invention is the tyrosine kinase analysis.Tyrosine residue in the tyrosine kinase phosphorylated substrate peptide.Existing tyrosine residue has fluorescence labeling again in the peptide substrate.In this was analyzed, a side end was selected as the antibody of phosphorylation residue, is attached to such as silica bead or hole lower surface with another end.Tyrosine kinase and have the fluorescence labeling peptide of tyrosine residue to be added in the hole.If this peptide of tyrosine kinase phosphorylation, the tyrosine of this phosphorylation will be attached on the antibody.Thereby fluorescence labeling is positioned the surface of antibody attachment.If tyrosine kinase does not have this peptide of phosphorylation, the fluorescence labeling on peptide will be scattered in the hole.Be bonded in the degree that surperficial fluorophor can be judged this peptide phosphorylation by mensuration.This analysis also can be used as on the special antibody of the fluorescent product that is acted on the fluorogenic substrate generation by enzyme.

In addition, can also carry out the analysis of living cells enzyme according to the present invention.Prior art is known to have the many technology that can analyze the enzymatic activity of living cells (to see Biolchem.Histochem.70,243 (1995), J.Fluorescence 3,119 (1993)), act on enzyme on the substrate that can produce fluorescence and (see Haugland, R.P. fluorescence probe and the chemical handbook of research, sixth version, the 10th chapter).In general, these are analyzed to use and enter cell is deposited in intracellular product then by desmoenzyme effect generation probe passively.Other substrate produces insoluble fluorescent product and is deposited on enzyme active sites.But the present invention's enzyme analysis activity degree and the site, accurate space of using such probe judgement enzymatic activity.Use the present invention to carry out the used probe of plurality of enzymes analysis and comprise phosphatase, ATP enzyme, 5` nucleotidase, DNA and RNA polymerase, peptase, proteinase, esterase and peroxidase, but be not limited only to this.

Enzyme activity assay can be according to top disclosed first or second exemplary embodiment and relevant graphical analysis scheme.

Morphology

Method of the present invention can also be used to carry out those need judge form cell or subcellular, comprises the analysis of aixs cylinder and organelle, but is not limited only to this.For carrying out such analysis, thereby by direct microinjection or use and by metabolism or to be changed the Premeabilisation of cells agent exposing cell that can be trapped in the research thing structure, fluorescence probe is imported the research thing for example in the structure of cell or organelle.If be used to living cells, this fluorescence labeling must be nontoxic and abiology activity.There are a lot of business-like suitable dyestuffs that can be used for this analysis (to see Haugland, R.P. fluorescence probe and research chemical handbook, sixth version, the 15th chapter), for example, comprise capillary continuous stream, neuronal cell connectivity, by the dyestuff transposition of slit connection, cell division and cytolysis and liposome fusion.In addition, these tacking agents can be used for following the tracks of the motion of the labeled cell in cultivation, tissue or not damaged organism.A lot of known technology with fluorescent tracer analysis of cells or subcellular fraction form or motion are arranged in the prior art, comprise and use biotin dextran conjugated body, fluorescein droplet or albumen and albumen conjugated body (to see Meth.Cell.Biol.29，153(1989)；Cytometry?21，230(1995)；Cell?84，381(1996)；Biolchem.Biophys.Acta?998，319(1989)；Cytometry?14，747(1993))。Various embodiment of the present invention have remarkable advantages when being used in the analysis of these types.The present invention allows the multiple parameter fast imaging with fabulous spatial resolution.

Nucleic acid

The present invention also can be used for carrying out the analysis of nucleic acid.The specific DNA analysis of benefiting from spatial resolution of the present invention and multi-wavelength imaging capability is fluorescence in situ hybridization (FISH).FISH is used for locating and judges relative content at cell, tissue, interphase nuclei and metaphase chromosome specific nucleic acid sequence, and J.27 the important techniques that is used for the clinical diagnosis and the assignment of genes gene mapping (sees Histo-chem, 4 (1995); Science 247,64 (1990); Trends Genet.9,71 (1993) and Science 250,559 (1990)).Multiple fluorescent hybridization probe is used to color fluorescence DNA and RNA hybridization technique (seeing Haugland, R.P. fluorescence probe and the chemical handbook of research, sixth version, the 8.4th chapter).Another kind of technology judges that by using AT or GC selective d NA dyestuff and nucleic acid to redye chromosome divides band.This technology is widely used in karyotyping and chromosome structure research (seeing Human Genet.57,1 (1981)).

Active oxygen

The present invention also can be used to analyze the level of various reactive oxygen species such as single peptide oxygen, superoxide and oxides of nitrogen.The importance of these reactive oxygen species just just is found (seeing BiplchemPharmcol 47,373 (1994), J.Cell Biol.126,901 (1994)).Most of the known physiological damage that is caused by active oxygen is to be caused by singlet oxygen (to see J.Photochem.Photobiol.11,241 (1991)) .Nitric Oxide (NO)), particularly, it is now know that, and it plays the numerator mediated effect of closing combination and (sees Current Biology 2 in comprising a lot of physiology courses of neurotransmission and blood pressure regulating, 437 (1995), J.Med.Chem.38,4343 (1995), Cell 78,919 (1994)).Prior art is known that uses round-about way to analyze NO.For example, under physiological condition, NO is oxidized to nitrite, this can be by monitoring it in the absorptance at 548nm place or use one to detect and (see Haugland to form certifiable fluorophor product with the probe of nitrite reaction, R.P. fluorescence probe and research chemical handbook, sixth version, the 21st chapter).

pH

The present invention also can be used to carry out and comprise in the mensuration cell or the analysis that pH changes in the acellular medium.The importance of internal pH role (is seen Cell Physiol.Biochem.2,159 (1992) comprising hyperplasia, apoptosis, fertilization, necrosis, multiple drug resistance, ion transport, lysosomal storage disease and early be realized already in various physiology such as old dementia and the pathologic process; J.Biol.Chem.270,6235 (1995); Biophys.J.68,739 (1995); J.Biol.Chem.270,19599 (1995); Cancer Res.54,5670 (1994)).The fluorescence probe that is used to analyze the pH variation in physiological range can buy (seeing Haugland, R.P. fluorescence probe and the chemical handbook of research, sixth version, the 23rd chapter).

Example

Here the invention of describing and declaring is by can more easily being understood by the common those of skill in the art of this area with reference to following example.These examples only are for several aspect of the present invention is described, and can not be interpreted as any limitation of the invention.

The transcription factor transposition

Cell grows in 96 orifice plates, fixes, become transcription factor protein, flushing, dye with 5 μ M Hoechst, 33342 damping fluids then with the antibody incubation of Texas Red mark.

Image is pressed 0.5x 0.5mm ²Rectangle observe, the pixel size in this rectangle is 1.08x 1.08 μ m ²Texas Red radiation is excited at the 568nm place, and the filtrator long by 600-nm detects.The Hoechst radiation is excited at the 364nm place, by the bandpass filter detection of 420-480-nm.The Image Acquisition time is 0.9 second.Every width of cloth image has～150 cells.

The image of the cell in inactive visual field obtained before fixing.Low in the strength ratio tenuigenin of Texas Red in nucleus.The generation antibody of Texas Red mark and a width of cloth composite diagram of the cell image that Hoechst 33342 dyes.

The image of the cell in inactive visual field obtained before fixing.Because colour code if nucleus is unsaturated, then is difficult to see tenuigenin.The generation antibody of Texas Red mark and a width of cloth composite diagram of the cell image that Hoechst 33342 dyes.

This data analysis is carried out in accordance with the following methods.A kind of binary representation of this Hoechst image produces by applying suitable threshold.Those values greater than threshold value are arranged to 1, and are arranged to 0 less than those values of threshold value.This is as original mask.Produce two masks of future generation then, one is to produce by the corrosion original mask, and another is by the expansion original mask and cuts original mask and produce to form the ring-type mask.Multiply by Texas Red radiation pattern the two-value mask that is corroded and these pixel summations rise and are used as measuring of the transcription factor that is labeled in this nucleus.Similarly, Texas Red radiation pattern multiply by ring-type mask and these pixel summations and rises be used as measuring of the transcription factor that is labeled in this tenuigenin.Activation degree uses nucleus and cytoplasmic intensity recently to assess.

The instantaneous calcium imaging that muscarinic receptor and voltage-gated channel stimulate

Figure 19 and 20 cell are to derive from neuroblastoma system.They are grown in reference fluid and imaging.These cellular expressions can be stimulated by carbachol, produce the muscarinic acetylcholine acceptor as second messenger's big intracellular calcium release.In addition, " L " calcium channel that these cellular expressions are valtage-gated, it is by extracellular K ⁺The cell membrane depolarization that the bigger variation of concentration causes stimulates, and suppressed by verapamil.

In general, can inspire image sequence by quick adding 100 μ L growth-promoting media reagent cell in the 100 μ L growth-promoting medias in 96 orifice plates.The stirring that is caused by this volume of liquid of adding causes the distortion that cell shape is little.This distortion is visible as first picture frame of calcium fluorescence transient change after adding that is distributed on each cell.

Shown between frame 1.2 seconds film at Figure 19.Image sequence is inspired by quick adding 100 μ M carbachols.Last piece image is a binary mask, is used for discerning and enumerate the fluorescence article in the image, and it produces the frame before injecting.Although image seems very fuzzy before the injection, it is very bright.This mask is used in each width of cloth image, each article in the sequence, and the intensity of integration is standardized into the preceding image of injection, is drawn into the table with respect to the time, shown in Figure 25 a.Mask is not processed into and covers whole cell.For example, article 1 may be more than a cell, but shows reactionless.Article 7 may be the cells of 2 coverings, show the time lag of first order reaction.

Figure 20 a-h is a select frame from a film, shows to cause valtage-gated by adding 50mM KCl " L " channel opener inspires the reaction of the neuroblast oncocyte of depolarization incident.Routine analyzer is described in the above as contact Figure 25 a.The result is presented at Figure 25 b.The acquisition of noting the increase of sensitivity is to use " cell is average " rather than " image averaging ".

The combination of living cells g protein coupled receptor

The image that Figure 21 a-c shows to 22a-c is that the living cells from 96 orifice plates obtains.These cells have been used the g protein coupled receptor transfection, and its native peptides part is known.Before the imaging, these cells are hatched 370 ℃ with natural unmarked part in the standard growth liquid that comprises 10% serum, and 20 minutes is 20nM fluorescence labeling part and 100nM LDS751 reaction 20 minutes afterwards, also is 37 ℃.Sample need not wash.

Image is 0.5x 0.5mm ², 1.08x 1.08 μ m ²G pixel.Fluorescent radiation is excited at the 488nm place, and by the bandpass filter of 45nm, central point detects at the 535nm place.The LDS751 radiation also is excited at the 488nm place, and by the bandpass filter of 40nm, central point detects at the 690nm place.The Image Acquisition time is 0.9 second.These cells are nearly～100,000 acceptor/cells, or 25 acceptor/m ²The film surface.

Figure 21 a is the image of cell after hatching with tagged ligand.Before imaging, do not carry out rinsing step.The actual change of receptor active is tangible.Some cells are in conjunction with considerably less part, and their video picture is submerged in background.Shown in Figure 21 b, by make the dyeing of mask, non-specific nucleic acid from the imaging of LDS 751 radiation, then the analysis in conjunction with active of cell becomes and is easy to one by one.This dyeing is not complete and homogeneous, but the major part of cell volume is all displayed.The covering of the binary mask of Figure 21 c that produces from the data threshold of Figure 21 b and receptors bind image produces the pseudo-chromatic graph of a receptor active.High activity is shown as yellow, and low activity is shown as orange red.

Figure 22 has shown the point on the titration curve of the 20nM tagged ligand of three image correspondences and unmarked part.This curve display is at Figure 22.Unmarked part is used AKi=3 ± 1 * 10 ¹⁰The M formula calculates.

Figure 23 a-d has shown the image of receptors bind on the different mammal clones.Figure 23 a is the image of the cell of hatching with 256nM Cy3 tagged ligand.In conjunction with active scope is visible.Figure 23 b shows the stack of the image that 1 μ M Hoechat, 33342 staining cells of Cy3 data and synchronization gain are examined.The latter can be used as the reliable sign of individual cells.Figure 23 c is the image of the cell of hatching with the 256nM Cy3 tagged ligand that has the unmarked part of 10 μ M, and Figure 23 d is the stack of the image that shows that these data and 1 μ M Hoechat, 33342 staining cells are examined.The effect of the liquid of replacing with unlabeled cells in Figure 23 c is clearly.High correlation between Figure 23 c and d has shown the validity with the volume recognizing cells of their eliminating.

Simulation is based on the receptors bind of silica bead

Figure 24 a-d has presented the image of the silica bead of Cy5 mark.This test is the simulated receptor binding analysis, and fluorescence labeling part wherein is attached to the film that is supported by droplet and limits on the acceptor.

The silicon droplet, diameter 4 μ m are coated with the biotin that gathers nitrogen propane and biotin NHS ester.The activity of silica bead absorbs the streptavidin of Cy5 mark of the silica bead of known quantity and analyzes by passing through of quantizing to remove from liquid with photofluorometer.Each silica bead is found and includes 1.3 * 10 ⁶Streptavidin molecule.By with the Cy5 mark of proper proportion and unlabelled streptavidin in advance mixing and and silica bead hatch together, make the silica bead bag by the Cy5 molecule of last known quantity.The load of silica bead equals 0.16,1.6 and the poly-nitrogen propane silica bead of 16fmole/200 μ g.Each silica bead has average 17,170 and 1700 marks respectively.Sample is placed into imaging in Costar 96 orifice plates.Cy5 647nm laser excitation, emitted fluorescence detects at the 40nm of 690nm bandwidth filter with the center.Scan image is in 1 μ m pixel, and about 0.7 second time obtained.

Load has the silica bead of 170 and 1700 molecules to be easy to be detected, and the 17-fluro silica bead can be recognized in the image that constitutes Figure 24.Those only load have the silica bead of unlabelled streptavidin can not produce visible intensity.

Claims (22)

1. confocal imaging system comprises:

A) be used to form the parts of the fasciculi exilis spot of electromagnetic radiation, this light beam is along the optical axis horizontal expansion of radiation propagation;

B) be used for this fasciculi exilis spot is directed into and focuses on first elongate area on first plane at article place, with be used for electromagnetic radiation with the emission of this article and direct into parts on one or more second elongate area, wherein each second elongate area is positioned on the second different plane with first planar conjugate;

C) at least one second conjugate planes, perhaps conjugate to the pick-up unit in the 3rd plane of at least one second conjugate planes, this pick-up unit comprises thereon the rectangular array that the detecting element of the electromagnetic radiation unanimity of launching from article is formed; With

D) be used for by move this fasciculi exilis spot with respect to article, or by coming the device of scan articles with respect to this fasciculi exilis spot mobile article, make the electromagnetic radiation of this emission be sent to the rectangular array that detecting element is formed, and by this pick-up unit and described scanning synchronised convert the electromagnetic radiation of described emission to a plurality of electric signal and represent;

E) slender space wave filter, the spatial filter of the elongated variable-width of choosing any one kind of them, it has a major axis of aiming at the second elongated district; With

F) be used on pick-up unit forming the parts of the image of second conjugate planes.

2. according to the confocal imaging system of claim 1, this fasciculi exilis spot that wherein directs into the electromagnetic radiation of article comprises two or more wavelength.

3. according to the confocal imaging system of claim 1, wherein article are positioned on the substrate noncontinuous surface, and this substrate has the continuous surface that extends with equidirectional with this noncontinuous surface, and described system also comprises a kind of focusing system, and this focusing system comprises:

A) the first focused beam acts spot of the electromagnetic radiation of first wavelength is arranged, the described first focused beam acts spot directs into noncontinuous surface via object lens, and is reflected via object lens by described noncontinuous surface;

B) have the second focused beam acts spot of the electromagnetic radiation of second wavelength, the described second focused beam acts spot directs into continuous surface via object lens, and is reflected via object lens by described continuous surface;

C) be used for parts that the radiation of first wavelength and the radiation of second wavelength that reflects via object lens are isolated;

D) first detecting device is used to detect the first focused beam acts spot that is reflected via object lens by described noncontinuous surface;

E) second detecting device is used to detect the second focused beam acts spot that is reflected via object lens by described continuous surface;

F) moving-member is used for moving object lens with respect to substrate, perhaps moves substrate with respect to object lens; With

G) controller that is connected with first and second detecting devices and moving-member, its middle controller is according to the first focused beam acts spot or second position of focused beam acts spot on substrate, in response to the signal operation moving-member from first detecting device or second detecting device.

4. according to the confocal imaging system of claim 1, wherein sweep unit comprises rotating optical element that is used for the inswept article of mobile fasciculi exilis spot or the movably objective table of placing article.

5. according to the confocal imaging system of claim 1, wherein be guided fasciculi exilis spot article, electromagnetic radiation and comprise one or more wavelength, and wherein second plane is single.

6. according to the confocal imaging system of claim 1, the wherein emission of the article in first elongated area of two or more wavelength of electromagnetic radiation from first plane, described system also comprises the parts that are used to isolate each wavelength that is launched, so that detect these segregate wavelength at least one by one or more pick-up units.

7. according to the confocal imaging system of claim 1, wherein article are positioned on the substrate noncontinuous surface, and this substrate comprises the continuous surface that extends with equidirectional with this noncontinuous surface, and described system also comprises a kind of focusing system, and this focusing system comprises:

A) direct into noncontinuous surface by object lens, and the focused beam acts spot of the electromagnetic radiation that reflects via object lens by described noncontinuous surface;

B) focused detector is used to detect the focused beam acts spot that is reflected via object lens by described noncontinuous surface;

C) moving-member is used for moving object lens with respect to substrate, perhaps moves substrate with respect to object lens; With

D) controller that is connected with focused detector and moving-member, its middle controller are adjusted moving-member according to the position of focused beam acts spot on substrate in response to the signal from focused detector.

8. according to the confocal imaging system of claim 3, wherein first and second wavelength are identical.

9. according to the confocal imaging system of claim 3 or 7, wherein said substrate is the microtitration orifice plate, and described noncontinuous surface is the end in a hole in the microtitration orifice plate.

10. according to the confocal imaging system of claim 6, wherein article are positioned on the noncontinuous surface of substrate, this substrate has the continuous surface that extends with equidirectional with this noncontinuous surface, and wherein two or more wavelength of electromagnetic radiation are launched from article, described system also comprises a kind of focusing system, and this focusing system comprises:

A) the first focused beam acts spot of the electromagnetic radiation of first wavelength is arranged, the described first focused beam acts spot directs into noncontinuous surface via object lens, and is reflected via object lens by described noncontinuous surface;

B) have the second focused beam acts spot of the electromagnetic radiation of second wavelength, the described second focused beam acts spot directs into described continuous surface via object lens, and is reflected via object lens by described continuous surface;

C) be used for parts that the radiation of first wavelength and the radiation of second wavelength that reflects via object lens are isolated;

D) first detecting device is used to detect the first focused beam acts spot that is reflected via object lens by described noncontinuous surface;

E) second detecting device is used to detect the second focused beam acts spot that is reflected via object lens by described continuous surface;

F) moving-member is used for moving object lens with respect to substrate, perhaps moves substrate with respect to object lens; With

G) controller that is connected with first and second detecting devices and moving-member, its middle controller is according to the first focused beam acts spot or second position of focused beam acts spot on substrate, in response to the signal operation moving-member from first detecting device or second detecting device.

11. confocal imaging system according to claim 6, wherein article are positioned on the substrate noncontinuous surface, this substrate has the continuous surface that extends with equidirectional with this noncontinuous surface, and wherein two or more wavelength of electromagnetic radiation are launched from article, described system also comprises a kind of focusing system, and this focusing system comprises:

A) direct into noncontinuous surface by object lens, and the focused beam acts spot of the electromagnetic radiation that reflects via object lens by described noncontinuous surface;

B) focused detector is used to detect the focused beam acts spot that is reflected via object lens by described noncontinuous surface;

C) moving-member is used for moving object lens with respect to substrate, perhaps moves substrate with respect to object lens; With

D) controller that is connected with focused detector and moving-member, its middle controller are adjusted moving-member according to the position of focused beam acts spot on substrate in response to the signal from focused detector.

12. use the confocal imaging system of substrate, wherein substrate comprises noncontinuous surface and the continuous surface that extends with equidirectional with this noncontinuous surface, described system comprises:

A) object lens via it, be focused on noncontinuous surface or will be guided at the first focused beam acts spot that is arranged in the electromagnetic radiation on the article of this noncontinuous surface;

B) have the second focused beam acts spot of the electromagnetic radiation of first wavelength, the described second focused beam acts spot directs into a focus on the noncontinuous surface via described object lens, and is reflected via object lens by described noncontinuous surface;

C) have the 3rd focused beam acts spot of the electromagnetic radiation of second wavelength that can be same as or be different from first wavelength, described three beams spot directs into a focus on the noncontinuous surface via described object lens, and is reflected via object lens by described noncontinuous surface;

D) be used for parts that the radiation of first wavelength and the radiation of second wavelength that reflects via object lens are isolated;

E) first detecting device is used to detect the second focused beam acts spot that is reflected via object lens by described noncontinuous surface;

F) second detecting device is used to detect the 3rd focused beam acts spot that is reflected via object lens by described continuous surface;

G) moving-member is used for moving object lens with respect to substrate, perhaps moves substrate with respect to object lens, so that control reflects the focus of bundle spot via object lens; With

H) controller that is connected with first and second detecting devices and moving-member, its middle controller is according to the second focused beam acts spot or the 3rd position of focused beam acts spot on substrate, in response to operating moving-member respectively from the signal of first detecting device or second detecting device;

I) slender space wave filter, the spatial filter of the elongated variable-width of choosing any one kind of them, it has a major axis of aiming at the second elongated district; With

J) be used on pick-up unit forming the parts of the image of second conjugate planes.

13. use the confocal imaging system of substrate, wherein substrate comprises noncontinuous surface and the continuous surface that extends with equidirectional with this noncontinuous surface, described system comprises:

A) object lens via it, be focused on noncontinuous surface or will be guided at the first focused beam acts spot that is arranged in the electromagnetic radiation on the article of this noncontinuous surface;

B) direct into noncontinuous surface via described object lens, and the focused beam acts spot of the electromagnetic radiation that reflects via object lens by described noncontinuous surface;

C) focused detector is used to detect the focused beam acts spot that is reflected via object lens by noncontinuous surface;

D) moving-member is used for moving object lens with respect to substrate, perhaps moves substrate with respect to object lens, so that control the focus of the focused beam acts spot that reflects via object lens; With

E) controller that is connected with focused detector and moving-member, its middle controller are according to the position of focused beam acts spot on substrate, in response to the signal operation moving-member from focused detector;

F) slender space wave filter, the spatial filter of the elongated variable-width of choosing any one kind of them, it has a major axis of aiming at the second elongated district; With

G) be used on pick-up unit forming the parts of the image of second conjugate planes.

14. according to the confocal imaging system of claim 12 or 13, wherein said substrate is the microtitration orifice plate, described noncontinuous surface is the end in a hole in the microtitration orifice plate.

15. be used for the method for item inspecting, comprise:

A) use the electromagnetic radiation of measuring the article emission from bioanalysis according to the confocal imaging system of claim 1;

B) use following process that a plurality of electric signal that produced by pick-up unit are divided into groups:

Accept a plurality of signals;

With a plurality of signals and threshold ratio;

According to the comparison of a plurality of signals and threshold value, set up a set corresponding to the reduced data value of these a plurality of signals; And

According to spatial relationship, the set of this reduced data value is grouped at least two groups corresponding to the part in a plurality of zones of the set of reduced data value.

16. be used for the method for item inspecting, comprise:

A) use the focusing image-forming system that contains with good grounds claim 13 or 14 to measure the electromagnetic radiation of the article emission from bioanalysis, wherein the electromagnetic radiation from the article emission is passed to pick-up unit and is converted into a plurality of electric signal;

B) use following process that a plurality of electric signal that produced by pick-up unit are divided into groups:

Accept a plurality of signals;

With a plurality of signals and threshold ratio;

According to the comparison of a plurality of signals and threshold value, set up a set corresponding to the reduced data value of these a plurality of signals; And

According to spatial relationship, the set of this reduced data value is grouped at least two groups corresponding to the part in a plurality of zones of the set of reduced data value.

17. be used for the method for item inspecting, comprise:

A) a fasciculi exilis spot of formation electromagnetic radiation, it is transverse to the optical axis extending of radiation propagation;

B) guide and focus on first elongated area of this fasciculi exilis spot in first plane of placing these article, and guiding from the electromagnetic radiation of article emissions to one or more second elongated area, wherein each second elongated area is positioned on the second different plane with first planar conjugate;

C) pick-up unit is put at least one second conjugate planes or be put into the 3rd plane of at least one second conjugate planes conjugation in, this pick-up unit comprises the rectangular array of being made up of detecting element, and the electromagnetic radiation from the article emission on this array is consistent; And

D) scan this article by this fasciculi exilis spot being moved with respect to article or article being moved with respect to this fasciculi exilis spot, make the electromagnetic radiation of emission be passed to the rectangular array of forming by detecting element, and be synchronized with the described a plurality of electric signal that convert the electromagnetic radiation of this emission to by this pick-up unit with scanning and represent.

18. according to the method for the item inspecting of claim 17, wherein article are positioned on the substrate noncontinuous surface, the continuous surface that this substrate has and this noncontinuous surface extends with equidirectional, and described method also comprises a kind of focus method, this focus method comprises:

A) the first focused beam acts spot that will have an electromagnetic radiation of first wavelength via object lens directs into noncontinuous surface, makes this first focused beam acts spot be reflected via object lens by described noncontinuous surface;

B) the second focused beam acts spot that will have an electromagnetic radiation of second wavelength that can be same as or be different from first wavelength via object lens directs into continuous surface, makes this second focused beam acts spot be reflected via object lens by described continuous surface;

C) radiation of first wavelength is isolated with the radiation of second wavelength that reflects via object lens;

D) detect the first focused beam acts spot that reflects via object lens by described noncontinuous surface with first focused detector;

E) detect the second focused beam acts spot that reflects via object lens by described continuous surface with second focused detector; With

F) according to the first focused beam acts spot or second position of focused beam acts spot on substrate, the signal in response to from first detecting device or second detecting device moves object lens with respect to substrate, perhaps moves substrate with respect to object lens.

19. according to the method for the item inspecting of claim 17, wherein article are positioned on the substrate noncontinuous surface, the continuous surface that this substrate comprises and this noncontinuous surface extends with equidirectional, and described method also comprises a kind of focus method, this focus method comprises:

A) via object lens the focused beam acts spot of electromagnetic radiation is directed into noncontinuous surface, make it reflect via object lens by described noncontinuous surface;

B) detect the focused beam acts spot that reflects via object lens by described noncontinuous surface with focused detector; And

C) according to the position of focused beam acts spot on substrate, the signal in response to from focused detector moves object lens with respect to substrate, perhaps moves substrate with respect to object lens.

20. the method according to the item inspecting of claim 17 also comprises the process of using the following, with the steps of a plurality of electric signal groupings that produce by pick-up unit:

A) accept a plurality of signals;

B) with a plurality of signals and threshold ratio;

C), set up a set corresponding to the reduced data value of these a plurality of signals according to the comparison of a plurality of signals and threshold value; And

D), the set of this reduced data value is grouped at least two groups according to spatial relationship corresponding to the part in a plurality of zones of the set of reduced data value.

21. use the focus method of substrate, wherein substrate comprises noncontinuous surface and the continuous surface that extends with equidirectional with this noncontinuous surface, described method comprises:

A) by the first focused beam acts spot of object lens guidings electromagnetic radiation, so that focus on noncontinuous surface or be arranged on the article of this noncontinuous surface;

B) have the second focused beam acts spot of the electromagnetic radiation of first wavelength via the object lens guiding,, and reflect via object lens by described noncontinuous surface so that focus on a focus on the continuous surface;

C) have the 3rd focused beam acts spot of the electromagnetic radiation of second wavelength that can be same as or be different from first wavelength via the object lens guiding,, and reflect via object lens by described continuous surface so that focus on a focus on the described continuous surface;

D) radiation of first wavelength is isolated with the radiation of second wavelength that reflects via object lens;

E) detect the second focused beam acts spot that reflects via object lens by described noncontinuous surface with first detecting device;

F) detect the 3rd focused beam acts spot that reflects via object lens by described continuous surface with second detecting device;

G) according to the second focused beam acts spot or the 3rd position of focused beam acts spot on substrate, in response to signal from first detecting device or second detecting device, move object lens with respect to substrate, perhaps move substrate, so that control reflects the focus of bundle spot via object lens with respect to object lens;

H) spatial filter by a kind of elongated variable-width carries out spatial filtering, and described spatial filter has a major axis of aiming at the second elongated district; With

I) image of formation second conjugate planes on pick-up unit.

22. use the focus method of substrate, wherein substrate comprises noncontinuous surface and the continuous surface that extends with equidirectional with this noncontinuous surface, described method comprises:

A) via the first focused beam acts spot of object lens guidings electromagnetic radiation, so that focus on noncontinuous surface or be arranged on the article of this noncontinuous surface;

B) via the focused beam acts spot of object lens guidings electromagnetic radiation to noncontinuous surface, feasiblely reflect via object lens by described noncontinuous surface;

C) detect the focused beam acts spot that reflects via object lens by noncontinuous surface with focused detector; And

D) according to the position of focused beam acts spot on substrate, the signal in response to from focused detector moves object lens with respect to substrate, perhaps moves substrate with respect to object lens, so that control the focus of the focused beam acts spot that reflects via object lens;

E) spatial filter by a kind of elongated variable-width carries out spatial filtering, and described spatial filter has a major axis of aiming at the second elongated district; With

F) image of formation second conjugate planes on pick-up unit.

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