CN101622522A - The method, system and the composition that are used for cell count and analysis - Google Patents

Abstract

The invention provides cheaply based on the system of imaging, be used for detecting, measure and/or the marker characteristic of counting biological sample, particularly blood sample.On the one hand, the present invention includes a kind of system that is used for a plurality of features of imaging sample, comprising: one or more light sources can produce the illumination beam that has the different wave length band separately continuously; But with a plurality of difference excitation labelings, can mark comprise the sample of a plurality of features, but so that variant feature by different difference excitation labeling marks.System of the present invention can further comprise the controller that functionally is associated with described one or more light sources, be used for illumination beam is directed to sample continuously, thereby but cause different difference excitation labelings continuously each be transmitted in light signal in the identical wavelength band; Optical system, can collect the light signal of this type of emission and on the photoresponse surface, form with sample be labeled the corresponding consecutive image of feature, thereby form its continuous image data set; And the one-trip container that is used for non-erythrocytic collection and optical analysis.

Description

The method, system and the composition that are used for cell count and analysis

Background technology

Point-of-care check (Point-of-care testing) and to search for effective biomarker be important theme in biomedical research, Holland et al for example, Curr.Opin.Microbiol., 8:504-509 (2005); Yager et al, Nature, 442:412-418 (2006); Frank et al, Nature Reviews Drug Discovery, 2:566-580 (2003); Sidransky, Nature Reviews Drug Discovery, 2:210-218 (2002).Two kinds of effort all are intended to improve the acquisition and the validity of health care, reduce its cost simultaneously.The point-of-care check is the analytical test that uses such device to carry out outside centralab, and described device is easy to be transported near the patient and moves under the condition in the open air, and need not the very personnel of specialty.In many acute nursing medical science and biological protection surveillance application, also need fast sample preparation and test to read, Raja et al for example, Clinical Chemistry, 48:1329-1337 (2002).

Biomarker is the objective determination of replying as normal biological process, pathogenic process or at the pharmacology that treatment is intervened and the feature of evaluation, Atkinson et al, Clin.Pharmacol.Ther., 69:89-95 (2001).Biomarker character, measure easiness and with the correlativity of the physiological status of target on alter a great deal Frank et al (more than quote) for example.Most of point-of-care devices are designed to measure the molecular biosciences mark extracted or directly to have found in biological fluid (for example blood) from sample or sample, Holland et al (more than quote).Measure on the cell marking in the care device at the scene great interest is arranged, but cell marking typically needs some form of imaging or fluid system to measure to carry out cell-specific, increased significant technological challenge for thus the mensuration of molecular labeling, Shapiro for example, Cytometry A, 60A:115-124 (2004); Shapiro et al, Cytometry A, 69A:620-630 (2006); Rodriquez et al, PLOS Medicine, 2 (7): e182 (2005); Janossy et al, Clinical Cytometry, 50:78-85 (2002); Toner et al, Annu.Rev.Biomed.Eng., 7:77-103 (2005); Deng.

The point-of-care check can be carried out on various sample types, not only comprises the sample from the individual organic body, for example medical science, animal doctor or plant sample, also comprise from all-environment sample, for example soil, water system, air-conditioning system, surface, public place, transportation system for example, or the like.In the medical sample, biological fluid, for example blood, saliva, lachrymal gland fluid, urine etc. are particularly suitable for the use that point-of-care is checked, because they are usually than easy obtain many of solid tissue.Can obtain from it biological fluid of cell or molecular labeling at this type of, as long as it is biologically relevant, blood is preferred sample, because blood is whole body, obtain easily, and comprise abundant and dynamic cell and molecule suspending liquid, the composition reflection health of described cell and molecule and the state of disease.Particularly, very big interest is arranged can counting on the non-erythrocytic specific subclass, described non-red blood cell is relevant with disease susceptibility, disease process, drug responsiveness etc., Guisset et al for example, Intensive Care Med., Epub (November 8,2006); Shakedet al, Curr.Cancer Drug Targets, 5:551-559 (2005); Madjid et al, J.Am.Coll.Cardiol., 44:1945-1956 (2004); Janossy et al (more than quote); Rodriquez et al (more than quote).Unfortunately, limited its widely used one or more following shortcomings for the current obtainable analyzer of this type of mark, comprise that complicated relating to separates and/or the preparation process of lysis, professional's participation, lack portability, expensive, lack sensitivity etc.

Consider above, several medical science and biological technical field will significantly be promoted owing to the acquisition of the technology that can carry out point-of-care operation, above-mentioned technology allow special in the biological fluid of for example blood the pair cell biomarker carry out measuring easily and neatly.

Summary of the invention

The invention provides cheaply based on the system (imaged-based system) of imaging, be used to detect, measure and/or count the feature that is labeled of biological sample, particularly blood sample.

In one aspect, the present invention includes a system that is used for a plurality of features of imaging sample, comprise following element: (a) one or more light sources, can produce the illumination beam that has the different wave length band separately continuously, (b) but a plurality of difference excitation labeling (differentially excitablelabel), the sample that can mark comprises a plurality of features, but so that variant feature by different difference excitation labeling marks; (c) controller that functionally is associated with described one or more light sources is used for illumination beam is directed to sample continuously, thereby cause that continuously but in the different difference excitation labelings each is transmitted in the light signal in the identical wavelength band; And (d) optical system, can collect the light signal of this type of emission and on the photoresponse surface (light-responsive surface) go up form with sample be labeled the corresponding consecutive image of feature, thereby form its continuous images data set.

In yet another aspect, but the present invention includes and a kind ofly be used for analyzing at the non-erythrocytic equipment of blood sample with a plurality of difference excitation labeling marks, this kind equipment comprises: (a) sample chamber, can the containment blood sample, and collect axle along light and have and stop a size that forms red blood cell light shield layer (light-obstructing layer); (b) a plurality of light sources, separately can be with illumination beam irradiation of blood sample with different wave length band, (c) be coupled to the controller of a plurality of light sources, be used for the illumination beam of each light source is directed to sample continuously, thereby cause that continuously but in a plurality of difference excitation labelings each is transmitted in the light signal in the identical wavelength band; (d) optical system can be collected the light signal of this type of emission and form the consecutive image corresponding with it on the photoresponse surface, thereby be formed the continuous images data set; Wherein the non-red blood cell in blood sample is counted by analyzing this type of continuous image data set.

In yet another aspect, the present invention includes a kind of one or more probe compositions that is used in a plurality of different cell analysis things of mark sample, the potpourri that comprises the specific probe of analyte, each probe can be bonded to different analytes specifically, wherein each probe is characterised in that: (a) in conjunction with the special binding compounds of pair cell analyte under the condition, and the optical markings that (b) is connected with binding compounds, the optical markings of variant probe has different excitation bands, and the optical markings of all probes is transmitted in the light signal in the same wavelength ranges.Preferably, this type of same wavelength ranges is separated with the excitation band of the optical markings of probe compositions.

In yet another aspect, the present invention includes a kind of disposable blood collection container (collection cuvette) that is used for non-erythrocytic optical analysis, described container comprises that (a) has the mixing chamber of the inlet that is used to admit whole blood sample, described mixing chamber further comprises dry reagent (driedreagent), dissolving and comprise probe compositions when this dry reagent can contact with whole blood sample, this probe compositions comprises the specific probe of a plurality of analytes, each probe can be specifically and the combination of non-erythrocytic different cell analysis thing, wherein each probe is characterised in that: (i) in conjunction with the special binding compounds of pair cell analyte under the condition, and the optical markings that (ii) is connected with binding compounds, wherein the optical markings of variant probe has different excitation bands, and the optical markings of all probes is transmitted in the light signal in the same wavelength ranges; And the sample chamber that (b) is connected with the mixing chamber fluid, so that the sample in the mixing chamber is sent to the sample chamber by capillary action, the sample chamber has optical transmission wall (transmissive wall) and the perpendicular size that equates basically with non-erythrocytic diameter, so that the light signal that is produced by the probe that is connected with the cell analysis thing is not covered by the red blood cell of sample.Preferably, select described size and between purpose cell and described optical transmission wall, form the erythroplastid light shield layer so that it stops basically.

In yet another aspect, the present invention includes a kind of disposable blood collection container that is used for non-erythrocytic optical analysis, wherein said container comprises: the sample chamber that (a) can receive whole blood sample, this sample chamber is placed in the body (body), and have at least one optical transmission wall and the perpendicular size that equates basically with non-erythrocytic diameter, so that the light signal that is produced by the probe that is connected with the cell analysis thing is not covered by the red blood cell of sample; And (b) reagent of the drying in the sample chamber, dissolving is to form probe compositions when itself and sample combination, this probe compositions comprises the specific probe of a plurality of analytes, each probe can be specifically and the combination of non-erythrocytic different cell analysis thing, wherein each probe is characterised in that: (i) in conjunction with the special binding compounds of pair cell analyte under the condition, and the optical markings that (ii) is connected with binding compounds, wherein the optical markings of variant probe has different excitation bands, and the optical markings of all probes is transmitted in the light signal in the same wavelength ranges.

In yet another aspect, the present invention includes a kind of equipment that is used to make by the sample imaging of a plurality of fluorescence labeling marks, this equipment comprises following element: (a) can shine one or more light emitting diodes of sample, each light emitting diode produces the illumination beam with different wave length band; (b) be coupled to the controller of light emitting diode, be used for the illumination beam of light emitting diode is directed to sample, thereby cause a plurality of fluorescently-labeled each sequential firing light signal; (c) optical system can be collected the light signal of emission and be formed the image corresponding with it with the generation view data on the photoresponse surface, and wherein optical system comprises and can catch a plurality of color cameras with wavelength optical signals.Preferably, described one or more light emitting diodes are a plurality of light emitting diodes, and described optical system produces a plurality of image data set, and each this type of group is corresponding with the light signal that produces in response to the irradiation by one of different described light emitting diode.

The invention solves the point-of-care system and be used for express-analysis medical science and environmental sample, the many costs and the efficient shortcoming of the prior art scheme when comprising blood, saliva, urine etc.Preferred implementation of the present invention is fit to various cellular components and/or the pathogen that low cost and effective detection and counting may exist well in whole blood, include but not limited to non-red blood cell, lymphocyte, for example CD3+ cell, CD4+ cell, CD8+ cell, haematozoon, for example malaria etc.

Description of drawings

Fig. 1 diagram illustration the optical system of using for the present invention.

Fig. 2 diagram illustration use to regulate the optical module system of excitation beam for LED.

Fig. 3 illustration be used to probe compositions of the present invention to select the principle of optical markings.

Fig. 4 A shows two kinds of fluorescently-labeled absorption curves that use in an embodiment, and these two kinds of fluorescence labelings have different excitation bands but can launch the fluorescence in same wavelength ranges basically.

Fig. 4 B shows three kinds of fluorescently-labeled absorptions and launching curve, and these three kinds of fluorescence labelings can sequentially be excited by three kinds of different excitation beams, and in being higher than the wavelength coverage of 650nm the emitting fluorescence signal.

Fig. 5 A-5C diagram illustration the embodiment of the sampling receptacle that uses for the present invention, this sampling receptacle is used for detecting non-red blood cell and/or other cell or the microorganism with analysis of whole blood.

Fig. 6 A is the image of the pearl that is placed on the phycoerythrin mark on the microslide of commercial acquisition.

Fig. 6 B shows the pearl concentration of proof mark and the data of the linear relationship between the pearl counting.

Fig. 7 A is the image from the cell of the anti-CD 4 antibodies double-tagging of the anti-CD 3 antibodies of using the APC-mark of whole blood and PECy5-mark.

Fig. 7 B shows in apc signal intensity with respect to the data from Fig. 5 in the X-Y scheme of PE signal intensity, and it shows three kinds of cell types, monocyte, CD4 ⁺T cell and CD4 ^-The difference of T cell bunch (cluster).

Fig. 8 A-8B shows the data that will compare with the counting that uses the flow cytometer acquisition from the complete blood count of the equipment of embodiment 1, and wherein said two devices is at the different sampling receptacle degree of depth (Fig. 6 A) with have the not isolabeling of 50 μ m (degree of depth) sampling receptacles (Fig. 6 B).

Fig. 9 shows from the data based on the check of pearl about proleulzin concentration.

Figure 10 diagram illustration the optical system of using for the present invention.

Embodiment

Unless indicate separately, practice of the present invention can be used to the routine techniques from molecular biology (comprising recombinant technique), cell biology, immunoassay technology, microscopy, graphical analysis, analytical chemistry, and these technology are in the scope of prior art.These routine techniquess include but not limited to the selection of detection, graphical analysis, irradiation source and the optical signal detecting assembly of fluorescence signal, mark of biological cell or the like.These routine techniquess and description can be found in such as following standard laboratory handbook: Genome Analysis:A Laboratory Manual Series (Vols.I-IV), Using Antibodies:A Laboratory Manual, Cells:ALaboratory Manual, PCR Primer:A Laboratory Manual, andMolecular Cloning:A Laboratory Manual (all coming from Cold SpringHarbor Laboratory Press); Murphy, Fundamentals of LightMicroscopy and Electronic Imaging (Wiley-Liss, 2001); Shapiro, Practical Flow Cytometry, Fourth Edition (Wiley-Liss, 2003); Herman et al, Fluorescence Microscopy, 2nd Edition (Springer, 1998); Foregoing is incorporated herein by reference in full for whole purposes.

The illumination beam that the invention provides by using different wavelength range sequentially shines system, the method and composition that cell, micelle, particle and/or analyte in the sample were measured and counted to sample, described different wavelength range corresponding to sample in analyte, cell or particulate directly or indirectly in conjunction with or the excitation band of the mark that is connected.After shining with this in proper order at every turn, collect light signal to form image, form image sets thus, wherein each width of cloth image comprises the analyzed view data of counting and/or measuring in order to colony that pair cell, particle and/or analyte are provided.In one aspect, utilize the non-overlapping basically a plurality of illumination beams of wavelength coverage.These a plurality of illumination beams are 2 to 6,2 to 4, perhaps between 2 to 3 the scope.A plurality of illumination beams can produce by the whole bag of tricks and the equipment that those of ordinary skills can utilize, and comprise by generations such as laser instrument, incandescent lamp and arc lamps.In one embodiment, illumination beam uses light emitting diode (LED) or similarly solid condition apparatus generation.Exemplary led light source comprises can (San Jose, the wavelength peak of CA) buying be the LuxeonTM LED of green (530nm), cyan (505nm), blue (470nm), reddish blue (455nm) from Lumileds Lighting LLC.Select the guidance of the specific LED of the present invention's use to obtain widely from technical literature, described technical information is such as Luxeon StarTechnical Data Sheet DS23 (Philips Lumileds Lighting Company, SanJose, 2006); Luxeon Star V Technical Data Sheet DS30 (LumiledsLighting, U.S., LLC, San Jose, CA, September 20,2004); Or the like.Usually, light source uses to produce the illumination beam of expectation wavelength coverage and intensity distributions with conventional optical filter and other optical modules.

I. optical system

The present invention can utilize various optical systems.Usually, this type systematic is provided for sequentially shining with different wavelength coverages one or more illumination beams of sample, be used to write down image gathering device, and the operation of control illumination beam and image gathering device is sequentially to collect the controller of image data set from the view data of illuminated sample.

In one aspect, the present invention relates to comprise the system of image gathering device, this image gathering device can excite the collaborative use of dye set with the difference that is connected to probe, and described difference can excite dyestuff that purpose cell, particle or analyte in the sample are had specificity.In other words, but this type systematic comprises the equipment that sample and sample with a plurality of difference excitation labeling marks are carried out imaging, and this equipment comprises following assembly: (a) a plurality of light sources, and each light source can both shine sample with the illumination beam with different wave length band; (b) controller is coupled to described a plurality of light source, be used for the illumination beam of each light source is directed to sample continuously, thereby but cause a plurality of difference excitation labelings continuously each be transmitted in light signal in the identical wavelength band; And (c) optical system, can collect the light signal of this type of emission and on the photoresponse surface, form consecutive image corresponding with it, form the continuous images data set thus.A kind of embodiment illustration in Fig. 1 of the said equipment.System (100) comprises some assemblies, comprises a plurality of light sources that are depicted as LED 1 (102) and LED 2 (104), be used for that sequential illumination is placed on that sample stage (116) goes up or within the viewing area (107) of sample (114); Image optics device (imaging optics) (106), be used for collecting light signal (109) in response to illumination beam (103) and (105) probe generation in sample and/or on the sample, and the signal guidance (111) that is used for collecting is to detecting device (108), detecting device (108) comprises the photoresponse surface such as CCD or cmos element, and light signal (109) forms image and from described photoresponse surface recording continuous images data set on described photoresponse surface.Preferably, the operation of system (100) is under the control of computing machine (110), computing machine (110) is used for the timing and the duration of (a) control illumination beam (103) and (105), (b) control the detecting device (108) that is used to collect view data and view data is sent to one or more databases, (c) analysis of image data is used for reading of read assembly with generation, and similar operations.Sample stage (116) can extensively change on the Design and Features performance, but sample need be placed usually in the geometrical construction on basic plane to meet the collection of a plurality of parallel optical signals and form image on detecting device.Preferably, the sample that is placed on the sample stage (116) is static, and is mobile or mobile; Even if perhaps there is motion, also be enough slow so that can collect consecutive image, described consecutive image can be compared during graphical analysis.Sample stage (116) can be included in conventional microslide, sample chamber or the container that uses in the microscopy, culture plate, microfluidic device etc.In one aspect, following will the description in more detail, sample stage (116) comprises the one-trip container that is designed to detect the non-red blood cell component in the whole blood.On the other hand, sample stage (116) comprises the container with sample chamber, and this sample chamber has as long as system (100) uses this type of container just to allow to measure the geometry of known volume.In one embodiment, this class sample chamber has the geometry on basic plane, wherein (a) bottom surface (or diapire) and end face (or roof) parallel to each other and (preferably) perpendicular to the minimum light path of image optics device (106), and (b) vertical range between roof and the diapire equates basically with the diameter of cell that will detect or particle.As long as this type of sample chamber is placed on (known or confirmable) viewing area (107), in the volume that cell or particle will be in known (maybe can determine), allow to measure the concentration of particle or cell thus." equating basically " about vertical range or size between sample chamber roof and the diapire refers in whole blood sample, is detectable from the non-red blood cell in the viewing area (107) or the light signal of particle.In other words, can be in the cell or the not transmission of shield light signal fully of the red blood cell between particle and the sample chamber roof (or other fragments (debris)) layer of mark.In one aspect, when white blood corpuscle is labeled and detect (for example CD4+ cell), the scope of vertical range is between 40 to 120 μ m, or between 50 to 100 μ m between roof and the diapire.The character of read assembly (112) can be shown to informative graphic user interface from simple numerical value, alters a great deal.In one embodiment, the read assembly (112) read by the counting that provides one or more predetermined cells or grain type of simple numerical value provides.In another embodiment, read the concentration that comprises one or more predetermined cells or grain type.And in another embodiment, read and comprise simple " be or non-" designator, whether be used to refer to or do not surpassed as yet the threshold level (for example, counting or concentration) of cell, particle or other analytes.

Utilizing LED to produce in the embodiment of illumination beam, can use optical module to regulate from the emission of selected LED, as illustrative pair of LED system among Fig. 2.The one LED (202) and the 2nd LED (206) have adjusting optical device (200) and (204) respectively, regulate optical device (200) and (204) and comprise diffusing globe (208), lens (210), bandpass filter (212) and lens (216) separately.The purpose of regulating optical device (200) and (204) is to provide the space to throw light on uniformly to sample (220).

Figure 10 illustrates that the present invention uses falls to penetrating (epi-illumination) optical system.LED (1000) produces illumination beam (1002), and illumination beam (1002) is by lens (1004) collimation and be directed to dichronic mirror (1006), then to object lens (1008).Light from illumination beam (1002) focuses on the sample (1010), and wherein fluorescence labeling is excited with the emitting fluorescence signal.To pass through dichronic mirror (1006) by the fluorescence signal guiding that object lens (1008) are collected, alternatively by emission optical filter (1012), arrive photoresponse surface (1014) subsequently, in this diagram, photoresponse surface (1014) is the personal digital assistant Zire72 Palm Pilot that can commercial buy, and it also comprises the display that is used for observing samples.Other illumination beam can add by add other dichronic mirror along light path between object lens (1008) and emission optical filter (1012).

II. difference can excite probe (differentially excitable probes)

On the other hand, the invention provides the composition that one or more the difference that is used in the multiple different analytes of mark sample can excite probe.Usually, probe compositions of the present invention comprises the potpourri of the specific probe of analyte, every kind of probe can both with different analyte specificity combinations, wherein every kind of probe be characterised in that (a) in conjunction with under the condition to the specific binding compounds of analyte (for example cell analysis thing), and the optical markings that (b) is connected with binding compounds, wherein the optical markings emission of the optical markings of every kind of different probe with different excitation bands and all probes is in the light signal in the same wavelength coverage.Generally, the aftermentioned wavelength coverage is not overlapping with any excitation band.Preferably, optical markings is the fluorescence labeling that can produce fluorescence signal, for example fluorescent dye.Yet the present invention also can use other fluorescence labeling, the plasma resonance particle that for example uses under the dark field illuminate condition.In one aspect, probe compositions of the present invention comprises that to multiple different analytes each has specific at least a probe.On the other hand, described multiple scope is between 2 to 8; Perhaps on the other hand between 2 to 4; Perhaps on the other hand between 2 to 3; And on the other hand, described multiple be at least 3 kinds; Perhaps scope is between 3 to 4.A key character of probe compositions of the present invention is: in the sample with the analyte of the different probe mark of composition, can by use optical markings to probe have every kind of probe of specific illumination beam continuous agitation optical markings and by sequence detection.Usually, when with the time interval of separating each illumination beam being guided to sample, this type of continuous agitation is not overlapping in time.In other words, illumination beam is guided to sample one at a time continuously.Preferably, in operation, from the light signal that respectively excites imaging on the photoresponse surface of detecting device, view data produces and is stored to be used for analysis from described photoresponse surface.When the light signal of probe is limited in a narrower wavelength coverage, because the image deterioration that the aberration of lens causes in the light path is just alleviated or eliminates.

The principle of operation of a kind of embodiment of probe compositions of the present invention illustration in Fig. 3, Fig. 3 shows the exciting and emission spectrum of optical markings of the composition of being made up of two kinds of probes of the present invention.First probe has the optical markings of use FRET (fluorescence resonance energy transfer) (FRET), wherein donor molecule has absorption or excitation spectrum (300) (dashed curve) and emission spectrum (302) (block curve), and acceptor molecule has and (302) overlapping absorption spectrum (304) (dashed curve) and emission spectrum (306) (block curve).Second probe has the fluorescence molecule with absorption spectrum (310) (dashed curve) and emission spectrum (312) (block curve) as optical markings.The maximum wavelength border of light signal scope is collected in dotted line (320) indication.Like this, need only sample quilt first illumination beam (330) irradiation of wavelength coverage as indicated, collect first light signal of forming by acceptor molecule emission spectrum (306) with first and second probe marks; Second light signal of forming by emission spectrum (312) as long as this type of sample by second illumination beam (340) of pointed wavelength coverage irradiation, just is collected in the same wavelength ranges.The exemplary D-A that is used for first probe is to (cyanine 3-allophycocyanin, Cy3-APC), the exemplary optics of second probe is labeled as Hua Jing 5, and (cyanine 5, Cy5) for flower cyanines 3-allophycocyanin.The exemplary optics marks packets that is used for three probe compositions is drawn together Hua Jing 7, and (cyanine 7, Cy7) (as donor that is used for first probe and acceptor), (APC is as donor for APC-Cy7, Cy7 is as acceptor, be used for second probe) and PE-Cy7 (PE is as donor, Cy7 is used for the 3rd probe as acceptor).

The further exemplary probe compositions that is used for double-tagging and three label probes illustrates respectively at Fig. 4 A (following description) and 4B.Fig. 4 B illustration three kinds of fluorescent dyes excite wavelength band with the relevant illumination beam of emission wavelength curve and probe compositions of the present invention.Dyestuff for many dinoflagellates phyllochlorin with excitation curve (422) and launching curve (428) (peridinin chlorophyll protein, PerCP), have phycoerythrin-Cy5 (PECy5) conjugate of excitation curve (424) and launching curve (430) and have excitation curve (426) and the allophycocyanin (APC) of launching curve (432).This type of dyestuff can excite in proper order by applying illumination beam, and described illumination beam is about 420-470nm for PerCP (434) wavelength coverage, is about 515-550nm for PECy5 (436) wavelength coverage, is about 590-640nm for APC (438) wavelength coverage.This type of illumination beam can be by LED, for example respectively by the indigo plant of Luxeon StarRoyal, green and blood orange (Red-Orange) LED generation.The fluorescence signal that is produced by probe uses bandpass filter (440) to separate with scattered light easily, and bandpass filter (440) only sees through the above light of about 650nm.Use conventional art, Hemanson for example, BioconjugateTechniques (Academic Press, New York, 1996), above-mentioned dyestuff is easy to be conjugated to for example binding compounds of antibody.

On the other hand, probe compositions comprises by the binding compounds of plasma resonance particle (PRP) mark.When being used for the dark field irradiation system, this type of probe compositions is particularly useful, thereby only collects from the scattered light of PRP.The PRP that is fit to use with probe compositions of the present invention discloses in following document: Schultz et al, Proc.Natl.Acad.Sci., 97:996-1001 (2000); Schultz etc., United States Patent (USP) 6,180,415; Prober etc., United States Patent (USP) 7,122,384; Or the like, be incorporated herein with for referencial use.In this embodiment, collect PRP so that its each the scattering of the biggest ground from the light of different illumination beams.

III is used for the container of whole blood assay

In one aspect of the invention, provide the one-trip container that uses for system of the present invention, to be used to carry out whole blood assay.In one embodiment, this type of container is used for counting the predetermined cellular blood species at predetermined, for example non-red blood cell; Thereby the cell count of this type of predetermined cell category or concentration can be used as to read and provide.Usually, one-trip container of the present invention comprises: the sample chamber that (a) can receive whole blood sample, this sample chamber is arranged in the body, and have at least one optical transmission wall and the perpendicular size that equates basically with non-erythrocytic diameter that will analyze, thereby the light signal that is produced by the probe that is connected with the cell analysis thing is not covered by the red blood cell of sample; And (b) reagent of the drying in the sample chamber, dissolving is to form probe compositions when itself and sample combination, this probe compositions comprises the specific probe of a plurality of analytes, each probe can be bonded on the non-erythrocytic different cell analysis things specifically, wherein each probe is characterised in that: (i) in conjunction with the special binding compounds of pair cell analyte under the condition, and the optical markings that (ii) is connected with binding compounds, wherein the optical markings of variant probe has different excitation bands, and the optical markings of all probes is transmitted in the light signal in the same wavelength ranges.Preferably, this type of one-trip container uses with aforesaid optical system, this optical system comprise the platform that is used for storage container so that vessel relevant for the fixed position of illumination beam and image optics device.Thereby this type of fixed position is aligned to as optical device and can collects from the light signal of container sample chamber.Be used to observe or measure the character of biological fluid, for example the design of the disposable specimen holder of blood parameters and be produced in the following document open: United States Patent (USP) 6,723,290; 6,869,570; 5,674,457; 5,200,152; 6,638,769; 4,088,448; Or the like, be incorporated herein by reference.

A kind of embodiment diagram of container of the present invention is illustrated in Fig. 5 A-5C.In one form, container (500) comprises body (501), and it can be glass, plastics or similar material, or its combination; And at least one sample chamber (502), it is connected with inlet (504) by passage (506).On the one hand, in order to use in whole blood assay, sample chamber (502) can hold scope from 5 to 100 μ L, or the sample liquid of 5 to 50 μ L volumes.Container (500) can also comprise the exhausr port (not shown) that is connected with sample chamber (502), enters the sample chamber and can not form back pressure (back pressure) to allow sample.Can also use and be used for sample pack into the alternative method of sample chamber (502), for example capillary action, suction, centrifugal force etc.A key character of container (500) is collected light signal for the volume that maybe can measure (512) from regulation, so that concentration determination can obtain from the view data of for example selected cell type.Volume (512) by the roof (514) of container (500) and the distance between the diapire (516) (for example, among Fig. 5 C 528) and the area of image optics device or field of view (507) limit, represent with the cone (508) and the direction (510) of collecting light signal.The key property of optical system of the present invention is in this embodiment: the pentrution of object lens is more than or equal to the distance (528 or 518) between roof (514) and the diapire (516), thus the light signal of in volume (512) all objects of collecting.Preferably, the light signal that roof (514) is fit to collect passes through, and substantially parallel with diapire (516).At another embodiment, container of the present invention can comprise other chamber, for example is used to hold reagent and/or is used for sample and this type of reagent were mixed before observing.In one aspect, container of the present invention further comprises dry reagent, for example comprise probe compositions, salt, buffering agent, the lytic reagent if desired time the etc., itself or directly be positioned over sample chamber (502), or, be included in the mixing chamber separately to activate before and and sample mix being sent to sample chamber (502) at another embodiment.

As mentioned above, the roof (514) of sample chamber (502) and the distance between the diapire (516) are important for the analysis of whole blood sample.If distance is excessive, (518) of Fig. 5 B for example, erythroplastid (520) may cover (526) and passes through light signal from what purpose cell type (522) produced so, and this type of cell may not be counted in this case, causes underestimating of cell number or concentration.According to the present invention, as shown in Fig. 5 C, the roof (514) of sample chamber (502) and the distance (518) between the diapire (516) equate basically with the diameter or the effective diameter of purpose cell type (522), thereby can't between purpose cell (522) and roof (514), form the shielding layer of erythroplastid, and from its light signal (524) all from the sample chamber (502) to the image optics device.In one aspect, sample chamber (502) have the distance (518) that equates basically with the pentrution of image optics device.In yet another aspect, sample chamber (502) have scope at 10 to 100 μ m, or 10 to 50 μ m, or the distance (518) in 20 to the 50 μ m.

Embodiment

In this embodiment, the imaging system of the present invention's use is constructed and is tested by cell or the particle counted in each sample.This system has according to the design shown in Fig. 1.Two different gray scale cameras (grey scale cameras) are used as detecting device.First is Sensovation Samba EZ140TC-cooled camera (cooled camera) (being lower than 20 ℃ of surrounding environment), has the 1392x1024 pixel, and pixel size 6.4um is square.Second camera is Point Grey Research Dragonfly2 industrial vision camera, has 1024x768 (4.65um) pixel.Use in the design of two kinds of imaging lens any.A kind of a pair of pairing spherical lens (doublet spherical lenses) that is designed to wherein has exciter filter to place therebetween.This system has high relatively N.A. (～0.33) and works good under the field of view up to about 2mm.Exceed this distance, the astigmatism distortion is remarkable, and increases apace with the image field increase.In order to solve this situation, use second lens devices.It is commercial camera lens (Nikon18-55mm f/3.5-5.6G ED AF-S DX Zoom), has the element of a compound non-spherical element (hybrid aspherical element) and an extremely low chromatic dispersion.These lens have good low distortion, and though pentrution preferably, and can surpass imaging the N.A. of it has lower～0.1 with can not detecting distortion under the field of view of 4mm.This decline of light collection efficiency is not enough to cause for detectable reduction on Cytometric any precision.Using this design of DragonFly2 camera and DX zoom lens is preferred configuration with regard to cost and image quality.

Led light source, or luminaire are equipped with himself exciter filter separately in lampshade.At propidium iodide (propidium iodide, PI) or under phycoerythrin/phycoerythrin (PE/PE) situation of one in front and one in back shining, Luxeon V Star Cyan (cyan) LED of lamp for having lambertian radiation (Lambertianradiation pattern), nominal peak wavelength 505nm (± 30nm spectral half width), 570mW under the nominal flux 700mA electric current.Exciter filter is the HQ510/50 optical filter available from Chroma.Excite for SYTO 17 or APC, lamp is Luxeon III Star Red-OrangeLED (blood orange LED, lambertian radiation), nominal peak wavelength 617nm (± 18nm spectral half width), nominal flux 1400mA electric current 600mW.Chroma HQ610/30 emission optical filter is used for blood orange light.LED uses being lower than under the maximum rated current.Particularly, unless otherwise noted, cyan (is used～75% flux peak) under 500mA, and reddish orange (is used～55% flux peak) under 700mA.As shown in Figure 2, from the LED element pattern of exciting light, (15 ° of angles are available from Edmunds Scientific) places before the LED with holographic diffuser for smoothly.Focus on long lens by a pair of 25mm light is accumulated in the sample imaging region.

Run through this research all the time, need to use software algorithm handle with analysis image identifying pearl, cell or other particle, and according to fluorescence intensity and particle diameter with they parametrizations.Image processing remains on relative minimum keeping the integrality of raw data, and only comprises the variation of zoomed image with the intensity of illumination on the compensating images.Particularly, it comprises the algorithm that uses local background convergent-divergent each pixel relative with average full figure background.The size of local background is modified to the suitable distance of representing with expection particle size range and every pixel of consideration purpose particle (image is amplified the back).For example, under the prevailing situation in this research, pearl diameter 3 is to 8um, cell dia 7 is to 15um, each pixel is represented the sample of 4um, and the window (striding 15 pixels) of discovery 60um is worked very good in this example system, does not consume too much CPU time simultaneously and is used for handling.After the image compensation illumination variation, another algorithm is tested and appraised the local strength's maximum value search purpose particle that satisfies statistical law, this statistical law is designed to avoid the false positive of the tactic pattern (for example, blood count plan line) from random noise, external source particle (dust etc.) and sample chamber.At first, algorithm is sought local maximum (bright pixels), this maximal value is at least 3 standard deviations on the ground unrest, by all ring of the pixel of at least 1.5 standard deviations encirclements on ground unrest, and has the ring (allowing the noise of statistics to change) that intensity subsequently reduces.Comprise and be used to reject other inspection that rational standard difference was identified and checked to two particles (duplicate particle).When identifying particle, another algorithm uses steepest decline fitting algorithm, the form of the Gauss equation of optimizing with fitting algorithm for this reason, simple (circle, the non-ellipse) Gaussian curve (Gaussian curve) of the best-fit of the intensity distributions of count particles.Along with at each particle record match statistics (fitting statistics) (squared residual and and card side), report the canonical parameter of height, radius, skew and X-Y position through identifying.Based on radius, height and the integrated intensity (volume under the Gaussian curve) of particle particle is classified (or " gating (gated) ") then, they are divided into different haemocyte groups.

Use the pearl of various special preparations to prove susceptibility, every pearl has a spot of border phycoerythrin (PE) molecule.These particles are by hatching BD α-Mouse-Ig κ Compensation Beads (the compensatory pearl that hatches with potpourri, Becton Dickinson p/n552843) prepares, wherein said potpourri has with the special antibody of the CD3 antigen (CD3-PE) of PE mark and to the special antibody of biotin labeled CD3 antigen (CD3-biotin), and two antibody have the stable every pearl of generation and have the very ratio of the pearl of the PE molecule boundary of low concentration.(Point Grey Research, Vancouver BC) make the pearl imaging under various time shutter and internal gain are provided with DragonFly2 CCD camera.For the number of the every particle PE of the pearl of gained molecule by determining with respect to PE Quantibrite pearl (BectonDickinson p/n 30495) convergent-divergent.In this research, the every particle of the darkest pearl goods (as shown in Fig. 6 A) produces 825 PE molecules, and can be from detecting high-gain (24dB) and the ground unrest under the medium time shutter (1s).The pearl of 825 PE molecules is the darkest test pearl, the adequate level that expression is higher than sensitivity detects (hundreds and thousands of fluorophores of every cell) to satisfy any cell count based on DNA, maximally related cell surface marker for example the CD3 on the T cell and T4 antigen (every cell dyes separately～150,000 and～50,000 PE molecule), and many other application, comprise that parasite detects and clinical detection based on pearl, for example at Morgan et al, Clinical Immunol., disclosed hemocytometer beads array (bead array) is incorporated herein by reference among the 110:252-266 (2004).

The dynamic range of electronic detectors (comprising this) is mainly set by the dynamic range of A-D converter, is reduced by signal noise then.The theoretical maximum dynamic range of 12-position A-D converter in single image of Dragonfly2 camera is set at 1-4096.Investigate the noisiness of relevant Dragonfly2 camera, wherein read noise and dark current are two chief reasons.These are measured by analysis image, and image is taken under 0 to 10 second time shutter in 0 to 24dB gain and scope.The image that the noise intensity substitution of calculating is produced under read noise that is provided with regard to each gain and dark current value.Noise increases with gain linearity, and for the actual range of condition determination, under 0dB gain 0.1s exposure, consuming 1.62 bits, under 24dB gain 10s exposure, consuming 6.24 bits.For best-case, this dynamic range with single image is reduced to 1-1334, for worst condition, is reduced to 1-54.Attention is for this system, and wherein sample keeps static before camera, and the effective dynamic range of instrument can CCD gain that many images change in real time (on the fly) simultaneously be provided with and the time shutter strengthens significantly by taking.Because intensity was directly proportional with time shutter and CCD magnification, this has increased by 2 to 3 orders of magnitude of obtainable dynamic range under physical condition.

Stride～PE Quantibrite pearl that the 100-dual intensity changes, be used to test this.The PEQuantibrite pearl is made up of the potpourri of the colony of four kinds of pearls, and each pearl of every kind all has specific PE molecule average.By this way, detecting device can be proofreaied and correct according to the PE molecule is absolute strength value.Like this, the every pearl of the brightest colony comprises (on average) 66408 PE molecules, and the every pearl of the colony of taking second place comprises 31779 PE molecules, is every pearl 8612 and 863 PE molecules then.PE Quantibrite pearl is provided with span (1-15X amplification) imaging with the time shutter and the gain of increase in a series of 0.1 to 20 second.Along with time shutter and gain increase, the dynamic range window moves to detect each pearl successively with the strength level that increases, and measures (Fig. 9) up to all integuments.With the method, the number of the slope of best-fit line and every pearl PE molecule is proportional, than only using single image to provide more accurate value.

First absolute counting of studying on this equipment that is applied as the cultured cell in the volume chamber.Excite and general transmit scope aspect according to the double-colored of this equipment, design is lived/dead detect (live/dead assay), wherein uses propidium iodide (PI) the dyeing dead cell of impermeability, the SYTO-17 dyeing all cells of impregnability.505nm (cyan) LED that PI is used in after 510/50 bandpass filter excites, and 617nm (blood orange) LED that SYTO-17 is used in after 610/30 bandpass filter excites.Used emission optical filter is 720/150 bandpass filter, its roughly comprise the PI emission spectrum 1/3rd and SYTO-17 emission spectrum 1/2nd (referring to Fig. 4 A, wherein illustration is as follows: PI absorption spectrum (400), PI emission spectrum (402, SYTO-17 absorption spectrum (404), SYTO-17 emission spectrum (406), first excitation wavelength range (408), second excitation wavelength range (410), and the wavelength coverage (412) of collecting light signal).

Three clones (A549, HeLa and U20S) and DNA QC particle (BectonDickinson p/n 349523 comprises chicken red blood cell nucleus and calf thymus nucleus) are used for this research.Because DNA dyeing is extremely bright with respect to cell surface marker or PE Quantibrite pearl, instrument susceptibility is owing to reducing the time shutter, gain or exciting LED electric current (all producing gratifying result) to reduce.Picture quality and fidelity are all very outstanding for PI and SYTO-17 (image 6A) dyeing.SYTO-17 can pass through the living cells film, and PI only can be by losing the cell membrane of part-structure integrality.Along with the membrane permeability increase of staining cell, nuclear PI dyeing increases.Therefore, PI dyeing can be from 1: 1 roughly with respect to the scope of the balance of SYTO-17 dyeing in these cells, most when the PI intensity that exceed several times among the DNA during PI replacement SYTO-17.

Living cells and dead cell are distinguished in the image of gained, counted to determine living cells and dead cell respectively, and total cell count.In a research, with recently by trypsinized and from culture flask the A549 cell of desorption, add the DMEM cell culture medium with the concentration of scope from 50 to 500 cells/uL.Each sample was hatched 10 minutes with 10uM SYTO-17+10uM PI, will be transferred to haemocytometer counting chamber from the aliquot of each sample then.Make sample imaging in haemocytometer counting chamber as mentioned above, with regard to living cells, dead cell and total cell count analysis image.Linear result outstanding (referring to Fig. 6 B), the R2 value of three kinds of countings are 0.99 or better.Do not carry out the optimization that image analysis algorithm or gating are handled, and be significantly from the background count of false-positive～25 cells/uL, though its can by analyze and the gating algorithm on improve and solve.

Said system is used for detecting and counting the CD4+ cell of blood sample.Use the blood sample of cracking, for seedless CD3-, CD4-and CD45-positive cell, the result is compared to using flow cytometer to want favourable many.CD3 and cd4 cell surface indicia have been used for all identifying the cell in the whole blood by adding fluorescently-labeled resisting-CD3 and anti-CD 4 antibodies respectively that shown in the data shown in Fig. 7 A-7B and 8A-8B, picture quality is outstanding.

The performance of said system further detects from the light signal of tradition based on the immune detection of pearl by counting and quantification.From BD Bioscience (San Jose, CA) the hemocytometer beads detects (cytometric bead assay, the pearl that being used to CBA) measured proleulzin (IL-2) combines with the IL-2 of several concentration, and use the scheme of manufacturer, Morgan et al for example, Clinical Immunology, 110:252-266 (2004) is with the anti--IL-2 antibody staining of mark.Replace with flow cytometry analysis from the signal of pearl be, make pearl imaging in said system of mark, then according to signal intensity with their countings with classify.The results are shown in Fig. 9.

Definition

Usually, unless otherwise noted, term used herein has the corresponding implication of conventional usage in the field related to the present invention, described field comprises analytical chemistry, biological chemistry, molecular biology, cell biology, microscopy, graphical analysis or the like, for example hereinafter the expression those: Alberts et al, Molecular Biology of the Cell, the 4th edition (Garland, 2002); Nelson and Cox, Lehninger Principles ofBiochemistry, Fourth Edition (W.H.Freeman, 2004); Murphy, Fundamentals of Light Microscopy and Electronic Imaging (Wiley-Liss, 2001); Shapiro, Practical Flow Cytometry, FourthEdition (Wiley-Liss, 2003); Or the like.

" analyte " is meant material, potpourri or the component in sample, detects it and whether exists or measure that it is quantitative.Analyte includes but not limited to modification after the genetic translation of part, protein of peptide, protein, polynucleotide, polypeptide, oligonucleotides, organic molecule, haptens, epi-position, biological cell, acceptor, glycoconjugate, vitamin, hormone or the like.The analyte relevant with single molecular entity can be a kind of incessantly, for example the different phosphorylation sites on same protein.

" antibody " or " immunoglobulin (Ig) " is meant protein natural or that pass through reorganization or the synthetic generation of chemical method, and it can specificly be bonded to specific antigen or antigenic determinant.Antibody is generally about 150,000 daltonian different four glycan albumen, is made up of with two identical heavy chains (H) two identical light chains (L).Every light chain links to each other with heavy chain by a covalent disulfide bonds, and the number of disulfide bond changes between the heavy chain of different immunoglobulin isotypes.Each heavy chain and light chain also have the interior disulfide bridge bond of chain of regular intervals.Each heavy chain at one end has variable region (V _H), be many constant regions then.Each light chain at one end has variable region (V _L), have constant region at the other end; The arrangement that is in line of first constant region of the constant region of light chain and heavy chain, the variable region of light chain and the variable region of the heavy chain arrangement that is in line.Constant region is not participated in directly and is made antibodies to antigen.Depend on the amino acid sequence of the constant region of its heavy chain, immunoglobulin (Ig) is divided into different classifications.The immunoglobulin (Ig) that five kinds of primary categories are arranged: IgA, IgD, IgE, IgG, and IgM, several in these can be further divided into subclass (homotype), for example, IgG ₁, IgG ₂, IgG ₃, IgG ₄, IgA ₁, and IgA ₂" antibody fragment " used herein, and phraseological variant are defined as the part of the complete antibody that comprises antigen binding site, or the variable region of complete antibody, wherein this part does not have the CH (be CH2, CH3 and CH4 depend on antibody morphism) in the Fc zone of complete antibody.The example of antibody fragment comprises Fab, Fab ', Fab '-SH, F (ab ') ₂With the Fv fragment; Dimer; Any antibody fragment (being called " single chain antibody fragments " or " single chain polypeptide " herein) of the polypeptide that primary structure is made of a kind of continuous amino acid residue of uninterrupted order, include but not limited to (1) strand Fv (scFv) molecule, (2) single chain polypeptide, it only comprises a variable region of light chain, or it comprises the fragment of three CDR of variable region of light chain, and do not have a relevant heavy chain part, and (3) only comprise the single chain polypeptide of a variable region of heavy chain, or it comprises the fragment of three CDR of variable region of heavy chain, and does not have relevant light chain part; And the polyspecific or the multivalence structure that form by antibody fragment.Term used herein " monoclonal antibody " (mAb) is meant the antibody that obtains from the colony of allo-antibody basically, and the independent antibody that promptly constitutes colony is identical, except the sudden change of a small amount of existence that might natural generation.Monoclonal antibody is a high special at single antigen site.In addition, and comprise that typically tradition (polyclone) Antibody Preparation at the different antibodies of different antigenic determinants (epi-position) is opposite, each mAb is at the single antigenic determinat on the antigen.Except that their antigentic specificity, the advantage of monoclonal antibody is that they can be next synthetic by the hybridoma cultivation, is not polluted by other immunoglobulin (Ig).The guidance of the production of used antibody and selection can easily be found in textbook and handbook in the immune detection, Harlow and Lane for example, Antibodies:A Laboratory Manual (Cold Spring Harbor LaboratoryPress, New York, 1988); Howard and Bethell, Basic Methods inAntibody Production and Characterization (CRC Press, 2001); Wild, editor, The Immunoassay Handbook (Stockton Press, New York, 1994), or the like.

" antigenic determinant " or " epi-position " be meant at molecule, the site of protein surface normally, and independent antibody molecule can be in conjunction with thereon; Usually protein has several perhaps how different antigenic determinants, and can with many not homospecific antibody responses.Preferred antigenic determinant is the phosphorylation site of protein.

" binding compounds " is meant the compound that can specificity be attached to certain target molecules.The example of binding compounds comprises antibody, agglutinin, nucleic acid, adaptive son etc., Sharon andLis for example, Lectins, 2 ^NdEdition (Springer, 2006); Klussmann, The AptamerHandbook:Functional Oligonucleotides and Their Applications (JohnWiley ﹠amp; Sons, New York, 2006).

" complex compound " used herein is the aggregate (assemblage) or the aggregate of the molecule that directly or indirectly is in contact with one another.In one aspect, " contact ", or more particularly, complex compound about molecule, or about " directly contact " of specificity or specificity combination, be meant two or more molecules enough near so that the interaction that complex molecule is propped up in attractive non-covalent interaction, this non-covalent interaction is Van der Waals force, hydrogen bond, ion and hydrophobic interaction for example, or the like.In this regard, the stable part of the complex compound of molecule is under testing conditions, and than the non-reunion of its ingredient, or non-complex status is more favourable on thermodynamics for complex compound.As used herein, " complex compound " typically refers to the stable aggregate of two kinds or above protein.In one aspect, " complex compound " is meant the stable aggregate of two kinds of protein, for example the antigen that combines with the antigenic determinant specificity of target protein.

" dry reagent " is meant detectable, for example buffering agent, salt, reactive compound, for example enzyme, co-factor or the like, or binding compounds, for example antibody, adaptive son or the like, its form with the dehydration preparation provide being intended to and improve storage life, transportation and processing easiness, improve and store or the like.Character, component and the production method of dry reagent alter a great deal, and the preparation of this type of material and to produce those skilled in the art be known, and this can be proved by following document: Franks etc., and United States Patent (USP) patent 5,098,893; Cole, United States Patent (USP) 5,102,788; Shen etc., United States Patent (USP) 5,556,771; Treml etc., United States Patent (USP) 5,763,157; De Rosier etc., United States Patent (USP) 6,294,365; Buhl etc., United States Patent (USP) 5,413,732; McMillan, U.S. Patent application 2006/0068398; McMillan etc., U.S. Patent application 2006/0068399; Schwegman et la (2005), Pharm.Dev.Technol., 10:151-173; Nail et al (2002), Pharm.Biotechnol., 14:281-360; Or the like, these documents are incorporated herein with for referencial use.Dry reagent includes but not limited to, solid and/or semi-solid particle, powder, tablet, crystal, capsule etc., and it can prepare in every way.In one aspect, Gan Zao reagent is the freeze-drying particle.The freeze-drying particle can have uniform composition, and wherein each particle has identical composition, and perhaps they can have different compositions, so that two kinds or abovely different types ofly have the different freeze-drying particles of forming and mix.The freeze-drying particle can comprise and be used for various detections and biochemical reaction, comprises the reagent of all or part of immune detection, the detection based on enzyme, zymolyte detection, dna sequencing reaction etc.In one aspect, freeze-drying particle of the present invention comprises excipient and at least a detectable.The freeze-drying particle can be with predetermined size and dimension preparation, and described predetermined size and dimension can be determined by the kind of the detection that will carry out, required reaction volume, required dissolution velocity etc.Dry reagent can comprise excipient, and it normally adds the inert substance of material to, to give material denseness or shape.Many to those skilled in the art excipient are known, can comprise many different chemical constitutions.The example that can be used for excipient of the present invention comprises carbohydrate, for example sucrose, glucose, trehalose, melezitose, dextrose and sweet mellow wine; Protein is BSA, gel and collagen for example; And polymkeric substance for example PEG and polyvinylpyrrolidone (PVP).The total amount of excipient can comprise single or multiple compound in the freeze-drying particle.In some embodiments, the kind of excipient is the factor of the hygroscopic amount of the dry reagent of control.Lower hydroscopicity can strengthen the integrality and the freezing tolerance of dry reagent.Yet from then on based composition is removed all water will have unfavorable effect to those reactive components, and described reactive component is protein for example, and it needs a certain amount of tie water to keep correct conformation.

" read " be meant measure and/parameter that can be exchanged into numeral or value that detects.Under some linguistic context, read the actual numerical value that is meant this type of collection or record data and represent.For example from the position and the fluorescence intensity that read as the signal that produces at each hybridization site place of microarray of the fluorescence intensity signals of microarray, therefore this type of is read and can deposit in every way or store, for example with the image of microarray, with digital form, or the like.

" sample " is meant some materials from biology, environment, medical science or patient source, wherein attempts to detect or measure target cell, particle, pearl and/or analyte.Term " sample " comprises biological sample, for example some blood, culture of microorganism or the like; Environmental sample, for example soil or water sample; Medical sample or sample, for example some blood or tissue; Or the like.Sample can comprise the sample in synthetic source.Biological sample can be animal (comprising the mankind), fluid, solid (for example ight soil) or tissue, and liquid and food and feed product and composition for example dairy products, vegetables, meat and meat by-products and refuse.Biological sample can comprise the material of taking from the patient, includes but not limited to culture, blood, saliva, celiolymph, hydrothorax, breast, lymph, phlegm, seminal fluid, puncture suction thing (needle aspirates) or the like.Biological sample can be from the domestic animal of various sections, and obtains in wild nature or the wild animal, includes but not limited to animals such as ungulate, bear, fish, rodent.Environmental sample comprises environmentally conscious materials (for example surface mass, soil, water and production piece), and available from the sample of food and dairy products processing instrument, unit, vessel, disposable or non-once article.These samples are not interpreted as restriction and are fit to sample type of the present invention.Term " sample " and " sample " replacedly use.

Be meant identification with " special " or " specificity " of another kind of molecule combination, contact and about a kind of molecule, this molecule nonrecognition basically simultaneously, contact or do not form complex compound with other molecule at two kinds of complex compounds that intermolecular formation is stable.On the one hand, " special " that combines with second molecule about first molecule is meant first molecule another molecule of identification and form the degree of complex compound with another molecule in reaction or sample, itself and the complex compound of second molecule formation maximum quantity.Preferably, this maximum is three ten at least percent.Usually, the molecule that relates to the specificity binding events has the zone on its surface, and/or has cavity under the situation of protein, causes specific recognition between the molecule that mutually combines.The example of specificity combination comprises that antibody-AI, enzyme-substrate interact, form duplex or triplex, receptor-ligand binding between polynucleotide and/or oligonucleotides, or the like.As used herein, be meant that about " contact " of specificity or specificity combination two kinds of molecules are enough near so that the interaction of weak non-covalent chemical interaction (for example Van der Waals force, hydrogen bond, base stacking interaction, ion and hydrophobic interaction an etc.) complex molecule.

Above-mentioned instruction is intended to illustrate the present invention, rather than is limited the scope of claim of the present invention by its details.Though preferred implementation of the present invention is described, but it will be apparent for a person skilled in the art that, can carry out various variations or improvement and not deviate from the present invention, and be intended to make appended claim to cover these type of changes and improvements that all fall into true spirit of the present invention and scope.

Claims (8)

1. system that is used for a plurality of features of imaging sample, described system comprises:

One or more light sources can produce the illumination beam that has the different wave length band separately continuously,

But a plurality of difference excitation labelings can mark comprise the sample of a plurality of features, but so that variant feature by different difference excitation labeling marks;

With the controller that described one or more light sources functionally are associated, be used for illumination beam is directed to sample continuously, but to cause that continuously in the different difference excitation labelings each is transmitted in the light signal in the identical wavelength band; And

Optical system, can collect the light signal of this type of emission and on the photoresponse surface, form with sample be labeled the corresponding consecutive image of feature, form its continuous images data set thus.

2. but one kind is used for analyzing at the non-erythrocytic equipment of blood sample with a plurality of difference excitation labeling marks, and described equipment comprises:

The sample chamber can the containment blood sample, and collects axle along light and have and stop a size that forms the red blood cell light shield layer;

A plurality of light sources separately can be with the illumination beam irradiation of blood sample with different wave length band;

Be coupled to the controller of a plurality of light sources, be used for the illumination beam of each light source is directed to sample continuously, thereby cause that continuously but in a plurality of difference excitation labelings each is transmitted in the light signal in the identical wavelength band;

Optical system can be collected the light signal of this type of emission and form the consecutive image corresponding with it on the photoresponse surface, thereby be formed the continuous images data set; And

Count non-red blood cell in the blood sample by analyzing the continuous images data set.

3. one or more probe compositions that is used in a plurality of different cell analysis things of mark sample comprises:

The potpourri of the specific probe of analyte, each probe can be bonded to different analytes specifically, wherein each probe is characterised in that: (a) in conjunction with the special binding compounds of pair cell analyte under the condition, and the optical markings that (b) is connected with binding compounds, the optical markings of variant probe has different excitation bands, and the optical markings of all probes is transmitted in the light signal in the same wavelength ranges, and the excitation band of described wavelength coverage and the optical markings of probe compositions separates.

4. disposable blood collection container that is used for non-erythrocytic optical analysis, described container comprises:

Mixing chamber with the inlet that is used to admit whole blood sample, described mixing chamber further comprises dry reagent, dissolving and comprise probe compositions when this dry reagent can contact with whole blood sample, this probe compositions comprises the specific probe of a plurality of analytes, each probe can be specifically and the combination of non-erythrocytic different cell analysis thing, wherein each probe is characterised in that: (a) in conjunction with the special binding compounds of pair cell analyte under the condition, and the optical markings that (b) is connected with binding compounds, wherein the optical markings of variant probe has different excitation bands, and the optical markings of all probes is transmitted in the light signal in the same wavelength ranges; And

The sample chamber, be connected with the mixing chamber fluid, so that the sample in the mixing chamber is sent to the sample chamber by capillary action, the sample chamber have the optical transmission wall and perpendicular basically with the size of non-erythrocytic equal diameters so that the light signal that is produced by the probe that is connected with the cell analysis thing is not covered by the red blood cell of sample.

5. container according to claim 4, wherein said size stop formation erythroplastid light shield layer between purpose cell and described optical transmission wall basically.

6. disposable blood collection container that is used for non-erythrocytic optical analysis, described container comprises:

Can receive the sample chamber of whole blood sample, this sample chamber is arranged in the body, and have at least one optical transmission wall and perpendicular basically with the size of non-erythrocytic equal diameters so that the light signal that is produced by the probe that is connected with the cell analysis thing is not covered by the red blood cell of sample; And

The reagent of the drying in the sample chamber, dissolving is to form probe compositions when itself and sample combination, this probe compositions comprises the specific probe of a plurality of analytes, each probe can be specifically and the combination of non-erythrocytic different cell analysis thing, wherein each probe is characterised in that: (a) in conjunction with the special binding compounds of pair cell analyte under the condition, and the optical markings that (b) is connected with binding compounds, wherein the optical markings of variant probe has different excitation bands, and the optical markings of all probes is transmitted in the light signal in the same wavelength ranges.

7. one kind is used to make the equipment by the sample imaging of a plurality of fluorescence labeling marks, and this equipment comprises:

Can shine one or more light emitting diodes of sample, each light emitting diode produces the illumination beam with different wave length band;

Be coupled to the controller of light emitting diode, be used for the illumination beam of light emitting diode is directed to sample, thereby cause a plurality of fluorescently-labeled each sequential firing light signal; With

Optical system can be collected the light signal of emission and be formed the image corresponding with it with the generation view data on the photoresponse surface, and wherein this optical system comprises and can catch a plurality of color cameras with wavelength optical signals.

8. equipment according to claim 7, wherein said one or more light emitting diode is a plurality of light emitting diodes, and described optical system produces a plurality of image data set, and each this type of group is corresponding with the light signal that produces in response to the irradiation by one of different described light emitting diode.

Images