CN102289065A - Auxiliary device and method for microscope system - Google Patents
Auxiliary device and method for microscope system Download PDFInfo
- Publication number
- CN102289065A CN102289065A CN201110128927XA CN201110128927A CN102289065A CN 102289065 A CN102289065 A CN 102289065A CN 201110128927X A CN201110128927X A CN 201110128927XA CN 201110128927 A CN201110128927 A CN 201110128927A CN 102289065 A CN102289065 A CN 102289065A
- Authority
- CN
- China
- Prior art keywords
- sample
- cell
- microscopic system
- servicing unit
- image
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/24—Base structure
- G02B21/26—Stages; Adjusting means therefor
Abstract
The invention relates to an auxiliary device and method for a microscope system. The auxiliary device is independent of the microscope system and includes: an article-placing platform for placing samples, the article-placing platform is not borne by the body of the microscope system; a displacement adjustment mechanism connecting the article-placing platform and used for adjusting the article-placing platform and relative position between the samples on the article-placing platform and the microscope system; and a signal processing unit electrically connected with the displacement adjustment mechanism, and used for controlling at least a part of adjusting motions of the displacement adjustment mechanism and an image acquisition device to acquire sample images. The auxiliary device is easily integrated into the microscope system for coordination work and separated from the microscope system, and prevents trouble of repeated dismounting and installation.
Description
Technical field
The present invention relates to be used for the servicing unit and the method thereof of microscopic system, it can adapt to the microscopic system of all size, can be so that analytic process robotization, summary and standardization, the error of avoiding the human operational error to bring, and can expand the function of microscopic system.
Background technology
Microscopy as bright field, dark field, phase contrast, fluorescence, differential interferometry, laser co-focusing microscopy etc., is widely used in surveying the micromechanism with amalyzing substances.In biology laboratory, microscopic system or device are used to observe cell, micro-structure and particle.
When utilizing microscopic system to analyze, traditional flow process is: the experimenter is placed into the sample that collects on the carrier (as microslide), and this transparent medium is placed on the objective table of microscopic system then.Next the experimenter perhaps takes the image of sample by regulating the zones of different that objective table two dimensional motion in the horizontal direction comes observing samples by camera.The position that past contact need be regulated the objective table in the vertical direction in this process obtains distinct image.This process is normally manually carried out, and therefore, when the quantity of the sample area of needs observation was very big, the amount that manual machinery is regulated also was very big.This dull operation often makes that the experimenter can't bear the heavy load, and through regular meeting because the accuracy of the instability of operating personnel's error or operation influence experiment.
In order to carry out sample observation by microscopic system expeditiously and to analyze, people often wish that whole analysis process can realize robotization.Two kinds of solutions are arranged in the prior art.
It at first is automated microscope.The objective table of this class microscope self can be realized one dimension, two dimension or the three-dimensional motion of sample by Mechatronic Systems.This series products costs an arm and a leg usually, and its subsidiary image processing software is often more common, can't satisfy the analytical work of specialization, as apoptosis, cell cycle, activity analysis etc.
Another kind of solution is the electricity driving displacement platform that is fixedly installed to the microscopic system body.A remarkable shortcoming of this class scheme is to be difficult for installing, and is difficult for after installing being separated with microscopic system, makes that microscopic system is difficult to restore to the original state.And, dismantle repeatedly and install also and can bring a lot of troubles.The remarkable shortcoming of another of this class scheme is to be difficult to adapt to the microscopic system of various different sizes.
In scientific research and producing, often need use self-reacting device, analyze as flow cytometer (Flow-Cytometer), cytoactive detector etc.The principle of flow cytometer is that cell suspension flows through the cell flow chamber, intersects vertically with single wavelength light beam (being generally laser), and cell is arranged in single file successively by the beam detection district under hydrodynamic action.Cell is often handled with fluorescent dye in advance, therefore produces fluorescence signal detecting under the beam excitation, and is detected device and catches.The physics of cell, chemical property can be analyzed by the processing to these optical signallings.This shows that the feature of flow cytometer is flowing of cell, stream of cells intersects with surveying light, and the automatic collection and the analysis of signal.Flow cytometer has been widely used in the research and the routine clinical work of aspects such as immunology, biological chemistry, biology, oncology and hematology.But because flow cytometer costs an arm and a leg, the operation and maintenance cost is very high, has therefore limited its application to a certain extent.
Summary of the invention
In order to overcome the shortcoming of above-mentioned prior art, one object of the present invention is to provide a kind of servicing unit and method that is used for microscopic system, thereby make microscopic analysis robotization, summary and standardization, the error of avoiding human operational error and instability to bring.
Servicing unit according to an embodiment of the invention is independent of this microscopic system and comprises the glove platform of placing sample, displacement governor motion, and signal processing unit.This glove platform be can't help the body load-bearing of this microscopic system.This displacement governor motion connects this glove platform, is used to regulate sample on this glove platform and the glove platform and the relative position between this microscopic system.This signal processing unit is electrically connected this displacement governor motion, is used to control regulating to small part of this displacement governor motion and moves, and control an image collecting device collected specimens image.
Householder method according to an embodiment of the invention, at first, the servicing unit that will be independent of microscopic system is placed into by the microscopic system.Sample to be detected then is placed on the glove platform.The displacement governor motion can be regulated the relative position between glove platform and the microscopic system, thereby makes an image collecting device can obtain the image of sample in different spatial.Can realize seeking automatically burnt and sample scanning by signal processing unit, and the image of sample is handled, the particle in the sample is discerned, counted and classifies.
The beneficial effect of servicing unit of the present invention and householder method is: the cost performance height, can realize the analytical work of specialization.And servicing unit of the present invention and method can adapt to the microscopic system of different size, and it is easy to be integrated into microscopic system collaborative work with it, and is easy to be separated with microscopic system, has avoided the trouble of dismantling repeatedly and installing.Simultaneously, the present invention further provides the apparatus and method of expanding common microscopic system function, made that the analysis means that in the past had only the self-reacting device (as flow cytometer) by costliness to implement is accomplished on common microscopic system.For example, microscopic system can be expanded on function by apparatus and method of the present invention is to detect cytoactive, green fluorescent protein (GFP) transfection efficiency and apoptotic analyser, or the like.
In one embodiment of the invention, comprise that is further sought a burnt subsystem automatically.
In one embodiment of the invention, further comprise a pump.
In one embodiment of the invention, further comprise a liquid acquisition device, in order to the fluid supply collection liquid sample outside this glove platform.
In one embodiment of the invention, this liquid acquisition device comprises fluid loculus and pump.The fluid loculus is positioned on this glove platform.This pump is in order to send into this fluid loculus from this fluid supply to this fluid loculus, and this pump is connected to this signal processing unit.
In one embodiment of the invention, further comprise at least one this image collecting device.
In one embodiment of the invention, image collecting device is the linear array images harvester.
In one embodiment of the invention, comprise that further at least one wave filter handles the light from sample, for example use at least 2 wave filters, for example use at least 8 wave filters, for example use at least 20 wave filters to handle light from sample.
In one embodiment of the invention, the detector that further comprises a kind of attribute of an illumination light of measuring microscopic system.
In one embodiment of the invention, the illumination of detector measurement microscopic system illumination light.
In one embodiment of the invention, further comprise at least one light source.
In one embodiment of the invention, seek Jiao automatically and comprise: sample is progressively moved in vertical direction, and to the sample imaging; A reference value of calculation sample image; And it is pairing as distinct image with the highest reference value in a plurality of reference values.
In one embodiment of the invention, seek Jiao automatically and comprise the Fourier transform that carries out image.
In one embodiment of the invention, further comprise sample is carried out a kind of among the following analysis: cytoactive analysis, Apoptosis analysis, the analysis of cell green fluorescent protein transfection efficiency, cell growth cycle analysis, lymphocyte analysis.
In one embodiment of the invention, further comprise the attribute of particle in the medium with a kind of graphical treatment of carrying out among following: scatter diagram, histogram.
In one embodiment of the invention, further comprise and surveying or algorithm that circle is surveyed is handled image utilization is oval.
In one embodiment of the invention, the mechanism of the relative position between adjusting sample or glove platform and the microscopic system can be divided into coarse adjustment and finely tune two and overlap independently mechanism.
In one embodiment of the invention, further comprise a preceding protruding end that is used for fixing distance between this device and the microscopic system.
In one embodiment of the invention, distance between the vertical direction center line of the light hole of glove platform and the optical axis of microscopic system can be adjusted between-150mm～150mm, and glove platform to the distance of the lower surface of base can be adjusted between-200mm～500mm.
In one embodiment of the invention, further comprise a carrier that holds sample, this carrier is common microslide, cover glass, or has the plain film that constant volume is measured loculus, or the fluid loculus.
In one embodiment of the invention, an image collecting device is a face battle array device, and has 10,000 effective unit of surpassing, and for example has 100,000 effective unit of surpassing, for example have 1,000,000 effective unit of surpassing, for example have 2,000,000 effective unit of surpassing.
In one embodiment of the invention, a light source is a kind of among mercury lamp, Halogen lamp LED, light emitting diode, laser diode, the solid light source.
In one embodiment of the invention, a wave filter comprises the optical filtering wave band of at least two separation, for example comprises the optical filtering wave band of at least 4 separation, for example comprises the optical filtering wave band of at least 8 separation, for example comprises the optical filtering wave band of at least 20 separation.
In one embodiment of the invention, use coarse adjustment and micro-adjusting mechanism to regulate relative position between sample and the microscopic system, and coarse adjustment and the initial position of micro-adjusting mechanism before each the measurement remain unchanged.
In one embodiment of the invention, can the three-dimensional that sample carries out horizontal two-dimension scanner uni vertical motion be moved.
In one embodiment of the invention, comprise that further the particle in the sample analyzes, wherein sample is medium, suspension, cell, cell culture, tissue, blood, body fluid etc., and particle is cell, biomarker (biomarker), protein, DNA, RNA etc.
In one embodiment of the invention, further comprise the dyeing processing of in advance sample being carried out common dye, fluorescent dye, fluorescence antibody or other fluorescent material.
Description of drawings
For above-mentioned purpose of the present invention, feature and advantage can be become apparent, below in conjunction with accompanying drawing the specific embodiment of the present invention is elaborated, wherein:
Fig. 1 is the ultimate principle figure of the servicing unit of a kind of form of the present invention.
Fig. 2 is the schematic diagram of the alternative servicing unit of the present invention, wherein uses pump and fluid loculus to collect sample.
Fig. 3 is the servicing unit detailed structure view of one embodiment of the invention.
Fig. 4 is the synoptic diagram of light hole on glove platform and the cantilever in one embodiment of the invention.
Thereby Fig. 5 in one embodiment of the invention is installed in the glove platform synoptic diagram of realizing on the displacement platform the two-dimensional scan of sample.
Fig. 6 is the synoptic diagram that utilizes detector that the microscopic system illumination light is measured in one embodiment of the invention.
Fig. 7 is the synoptic diagram that utilizes automatic light source illumination sample and carrier in one embodiment of the invention.
Fig. 8 is the reflection cell fluorescence color of software program drafting in one embodiment of the invention and the scatter diagram of intensity attribute.
Fig. 9 is the statistic histogram of the reflection cell cycle distribution that software program is drawn in one embodiment of the invention.
Figure 10 is the method flow of one embodiment of the invention.
The numbering explanation
1. microscopic system
2. image collecting device
3. microcobjective
4. glove platform
5. carrier
6. signal processing unit
8. motor
10. motor
11. medium to be measured
12. fluid loculus
13. pump
18. knob
110. vertical displacement platform
120. horizontal shift platform
125. one dimension displacement platform
130. floor
135. cantilever
150. hand-operated lifting platform
160. preceding protruding end
170. base
180. slide block
185. screw rod knob
190. handgrip
200. housing
220. the illumination light of microscopic system
225. illumination light
240. detector
260. light source
Embodiment
According to design of the present invention, at first, the servicing unit that will be independent of microscopic system is placed into by the microscopic system.Sample to be detected then is placed on the glove platform.The load-bearing of this glove platform is not the body by microscopic system, but by this servicing unit itself.A mechanism of servicing unit can regulate the relative position between aforementioned glove platform and the microscopic system, thereby makes image collecting device can obtain the image of sample in different spatial.Can realize seeking automatically burnt and sample scanning by signal processing unit, and the image of sample be handled, the particle in the sample is discerned, counted and classifies by software program.
According to the present invention, this independent device is placed to by the microscope.Then sample is placed on the support that is subordinated to this device.Then this sample can be changed by this device with respect to microscopical position.In addition, can use a signal receiver and/or signal analysis device to absorb and/or handle the image of microscopy.
Fig. 1 is the ultimate principle figure of the servicing unit of a kind of citation form of the present invention.The servicing unit of this form comprises glove platform 4, displacement governor motion and the signal processing unit 6 of placing sample.This servicing unit is independent of microscopic system 1, and it is other to be placed to microscopic system 1 in use, with microscopic system 1 without any fixedly connected.Then on the testing sample suppressed by vector 5.Sample can be medium, suspension, cell, cell culture, tissue, blood, body fluid etc.Carrier 5 can be common microslide and cover glass, also can be to have plain film or the fluid loculus (flow cell, flow chamber) that constant volume is measured loculus.Then, carrier 5 also is that sample is placed on the glove platform 4 of servicing unit.The load-bearing of glove platform 4 is not the body by microscopic system, but by servicing unit itself.
The displacement governor motion has the displacement regulating power of horizontal direction and vertical direction by motor 8 and 10.Particularly, can drive the displacement platform of a vertical direction to change sample and microscopic system 1 relative position (direction in vertical direction by signal processing unit 6 control motors 8
), the vertical range between sample and the microcobjective 3 in other words, thus can be so that sample accurately focuses on.Can also control the displacement platform that motor 10 drives horizontal direction by signal processing unit 6 sample is carried out one-dimensional scanning, promptly change sample and microcobjective 3 relative position (direction in the horizontal direction
), make zones of different imaging on image collecting device 2 of sample.So just realized the two dimensional motion of sample with respect to microscopic system.Signal processing unit 6 control image collecting devices 2 obtain image, and the image that is produced is handled automatically, thereby can analyze the particle in the sample.Wherein this sample is at least one in organizing with next: medium, suspension, cell, cell culture, tissue, blood, body fluid; This particle is at least one in organizing with next: cell, biomarker (biomarker), protein, DNA, RNA.
Fig. 2 is the another kind of citation form of servicing unit of the present invention.Liquid acquisition device, displacement governor motion and signal processing unit 6 that the servicing unit of this form comprises the glove platform 4 of placing sample, is made of pump 13 and fluid loculus 12.Fluid loculus 12 is placed on the glove platform 4.By a pump 13 that is connected with signal processing unit 6 medium 11 samplings to be measured being sent to carrier then is in the fluid loculus 12.
The displacement governor motion has the perpendicular displacement regulating power.Specifically, drive the displacement platform of a vertical direction to change sample and microscopic system 1 relative position (direction in vertical direction by knob 18 of manual rotation
), the distance between sample and the microcobjective 3 in other words, thus can be so that sample accurately focuses on.Next utilize signal processing unit 6 to form image with image collecting device 2, thereby obtain a series of images signal with the medium that flows in the particular time interval convection cell loculus 12.Utilize 6 pairs of images that obtained of signal processing unit to handle then, can analyze the particle in the medium automatically.
Fig. 3 is the detailed structure view of the displacement governor motion of one embodiment of the invention, and this displacement governor motion can be applicable in the servicing unit shown in Figure 1. Motor 8 and 10 are installed in the housing 200.They are respectively applied for and drive vertical displacement platform 110 and horizontal shift platform 120.In fact, motor 8 and vertical displacement platform 110 constitute a vertical electricity driving displacement platform, and motor 10 and horizontal shift platform 120 constitute a horizontal electric displacement platform.Floor 130 is installed on horizontal shift platform 120.Cantilever 135 and glove platform 4 have light hole, are installed on the floor 130, as shown in Figure 4.
The mechanism that regulates the relative position between sample or glove platform 4 and the microscopic system can be divided into coarse adjustment and overlap independently mechanism with fine setting two.Motor 8 and 10 and vertical displacement platform 110 and horizontal shift platform 120 constitute micro-adjusting mechanisms, be used for the fine setting of glove platform 4 with respect to the microscopic system position.Coarse adjustment mechanism is made up of a manual displacement platform and a hand-operated lifting platform 150.Hand-operated lifting platform 150 is used for the coarse adjustment of glove platform 4 vertical direction positions.Rotary handle 190 can make housing 200 vertically move together with glove platform 4 on a large scale.The manual displacement platform is made up of adjusting screw(rod) knob 185, slide block 180 and base 170.Adjusting screw(rod) knob 185 can make slide block 180 move on base 170, moves horizontally on a large scale together with glove platform 4 thereby drive housing 200.Preceding protruding end 160 is used for fixing the relative distance of device of the present invention and microscopic system.By the coarse adjustment function, make servicing unit can be adapted to the microscopic system of different size.In the present embodiment, micro-adjusting mechanism is electronic, and coarse adjustment mechanism is manual.In another embodiment of the present invention, fine setting, coarse adjustment mechanism all are electronic.
In one embodiment of the invention, because the length of preceding protruding end 160 is fixed, slide block 180 all is fixed with respect to the position of base 170 and the height of hand-operated lifting platform 150, i.e. coarse adjustment mechanism initial position fixedly remains unchanged.
Simultaneously, set by software program, vertical displacement platform 110 was in identical position with horizontal shift platform 120 before each the measurement, and promptly the fine adjustment mechanism initial position fixedly remains unchanged.Therefore the initial space position of glove platform 4 when each the measurement all remains unchanged.
In one embodiment of the invention, distance between the center line of the vertical direction of glove platform 4 center light holes and the optical axis of microscopic system can be adjusted between-150mm～150mm, and glove platform 4 to the distance of the lower surface of base 170 can be adjusted between-200mm～500mm.
In a preferred embodiment of the present invention, distance between the center line of the vertical direction of glove platform 4 center light holes and the optical axis of microscopic system can be adjusted between-75mm～75mm, and glove platform 4 to the distance of the lower surface of base 170 can be adjusted between 100mm～300mm.
In one embodiment of the invention, glove platform 4 is positioned on the electricity driving displacement platform 125, and the direction of motion of the horizontal shift platform in the direction of motion of this displacement platform and the housing is perpendicular, as shown in Figure 5.Like this, just can realize horizontal two-dimension scanning to sample.Add the motion on the vertical direction, realized that in the present embodiment the three-dimensional of sample moves.
In one embodiment of the invention, use detector 240 to measure a kind of attribute of the illumination light 220 of the microscopic system of passing light hole, as shown in Figure 6.In a preferred embodiment of the invention, use detector 240 to measure the illumination of the illumination light 220 of microscopic system.The illumination of system illumination is consistent when using this brightness value can control each the measurement.
In one embodiment of the invention, use throw light on carrier 5 on the glove platform of light source 260 emissive lighting light 225, as shown in Figure 7.In one embodiment of the invention, illumination light 225 is directional lights.In one embodiment of the invention, illumination light 225 is convergent beams.In one embodiment of the invention, light source 260 adopts mercury lamp.In one embodiment of the invention, light source 260 adopts Halogen lamp LED.In one embodiment of the invention, light source 260 adopts light emitting diode (LED).In one embodiment of the invention, light source 260 adopts laser diode (LD).
In the fluorescence microscopy system, often use wave filter to select required spectrum frequency range.General fluorescence microscopy system all carries filter module or module satisfies different measurement needs.If microscopic system is not from band filter, apparatus of the present invention then additionally provide wave filter to use to microscopic system.In one embodiment of the invention, use at least one wave filter to handle light, for example use at least 2 wave filters, for example use at least 8 wave filters, for example use at least 20 wave filters to handle light from sample from sample.
In one embodiment of the invention, an optical filtering wave band that comprises at least two separation among the wave filter for example comprises the optical filtering wave band of at least 4 separation, for example comprises the optical filtering wave band of at least 8 separation, for example comprises the optical filtering wave band of at least 20 separation.
Automatically seek Jiao
Owing to reasons such as machine error and vibrations, when testing sample moves to diverse location with respect to microscopic system in the horizontal direction via motor and displacement platform, the phenomenon of out of focus might appear.That is to say that sample departs from the focussing plane of microcobjective, thereby on image collecting device, can't obtain distinct image.In order to address this problem, to have developed in one embodiment of the invention and sought burnt function automatically.Its principle of work is: utilize motor-driven displacement platform to make sample carry out progressively moving in the certain limit of vertical direction.Each goes on foot all to the sample imaging, and utilizes a reference value of signal processing unit computed image.These values are compared, pairing with the highest reference value as distinct image.In this way can so that the focusing process carry out automatically.This shows that electricity driving displacement platform, image collecting device and signal processing unit be actual have been constituted one and seek burnt subsystem automatically.
A kind of computing method of a reference value of foregoing image are that image is carried out Fourier transform and calculates the power spectrum of image.Fourier transform can adopt discrete Fourier transform (DFT) or fast fourier transform.Owing to do not consider the directivity of power spectrum characteristic, therefore the given spatial frequency of power spectrum is carried out radially average, and with it as the reference value of seeking Jiao automatically.
Figure 10 illustrates the basic procedure of the householder method of various embodiments of the present invention.The embodiment that will describe is haply based on this flow process below.
Detect yeast activity
In the present embodiment, servicing unit of the present invention is applied to the detection that microscopic system can realize the fluidic cell ceremony, the activity of yeast cells is analyzed.
The microscopic system of present embodiment is a fluorescent microscope, and signal processing unit is a computing machine, and image collecting device is effective element number or pixel value greater than 10,000 CCD area array cameras.In a preferred embodiment of the invention, used effective element number to surpass 100,000 CCD area array cameras.In a preferred embodiment of the invention, used effective element number to surpass 1,000,000 CCD area array cameras.In the preferred embodiments of the present invention, used effective element number to surpass 2,000,000 CCD area array cameras.
With reference to figure 1.At first servicing unit of the present invention is placed into by the fluorescent microscope.Yeast cells uses SYTO9 and two kinds of fluorescent dyeings of PI to handle in advance, is placed into then on the carrier 5.As shown in the figure, also be that testing sample is placed on the glove platform 4 of apparatus of the present invention with carrier 5.Next the displacement platform that drives vertical direction by computer control motor 8 is sought Jiao automatically, makes that the cell in the sample forms clearly image in the CCD camera, and wherein Lv Se cell is a living cells, and red cell is a dead cell.Computing machine obtains this image and carries out analyzing and processing.Next computing machine sends instruction to motor 10, makes the displacement platform drive testing sample of horizontal direction move a specific distance (being 0.2mm in the present embodiment).Move horizontally finish after, computing machine is controlled motor 8 once more and is sought Jiao automatically, obtains image and carries out image analysis processing.In the present embodiment, the computing machine repetition is sent for 8 times and is moved horizontally instruction, thereby obtains 9 width of cloth images of sample zones of different.
This shows, cell is owing to moving of carrier realized " flowing " in device of the present invention, stream of cells and probe microscope light intersect, and optical signalling is obtained automatically and is analyzed, this principle of work with flow cytometer is consistent, has therefore just realized the working method of flow cytometer like this.
The flow process of picture signal process software is as follows: at first utilize the oval algorithm of surveying that image is handled, find out the ellipse in the image, with the outline of these ellipses as cell.Then the pixel value of R in the oval scope (red) and G (green) passage is averaged respectively, as the metric of judging this cell color.With G, R passage mean value is coordinate, then can draw out scatter diagram as shown in Figure 8.Scatter diagram is a kind of common method of the attribute of cell being carried out graphical treatment.Corresponding decision rule (for example, if G passage mean value＞R passage mean value judges that cell is a living cells, otherwise being dead cell) is set in software program, just can tells living cells and dead cell by right area.In one embodiment of the invention, the algorithm that adopts circle to survey is handled image, finds out circle in the image as the outline of cell.
Can only obtain picture signal after above-mentioned steps is finished and concentrate the par of cell, comprise what cells in promptly average every width of cloth image.This numerical value multiply by a transformation factor, comprises what cells in promptly every ml cells suspension, promptly can be converted to the concentration of cells in sample, comprises what cells in promptly every ml cells suspension.This transformation factor can pre-determine by the contrast experiment with cell counter (hemocytometer).
Therefore the active estimated value of cells in sample can be calculated by following formula:
Cytoactive=(total cell concentration-dead cell concentration)/total cell concentration
Perhaps
Cytoactive=(total cell quantity-dead cell quantity)/total cell quantity
Cell culture in the present embodiment also can only be handled with the single fluorescent dye of PI in advance.Like this, only can show red dead cell in the image.Same, at first utilize the oval algorithm of surveying that image is handled, find out the ellipse in the image, the R in the RGB passage (red) passage is averaged to the pixel value in the pairing ellipse of cell then, as the metric of this cell fluorescence intensity.A threshold value is set in software program, and the cell that is higher than this threshold value is identified as dead cell.
Obtain picture signal after above-mentioned steps is finished and concentrate the par of dead cell.This numerical value multiply by a transformation factor, comprises what dead cells in promptly every ml cells suspension, promptly can be converted to the concentration of dead cell in the sample.Because living cells can not dyeed by PI, therefore in fluoroscopic image, can not show, cause viable cell concentrations or total cell concentration to can not determine by above-mentioned steps.If will determine cytoactive, promptly living cells accounts for the ratio of total cell, fluorescent microscope need be transformed into bright field illumination mode (general fluorescent microscope all has the bright field illumination system) total cell concentration is detected.Like this, only need the ellipse in the image is discerned quantity and the concentration value that counting just can obtain total cell.
Detect Apoptosis
In the present embodiment, device of the present invention is used for the detection that microscopic system can realize the fluidic cell ceremony, the apoptosis of Jurkat cell is detected.
With reference to figure 2.The microscopic system of present embodiment is a fluorescent microscope, and signal processing unit is a computing machine, and image collecting device is a CCD line-scan digital camera, and the pump of use is a peristaltic pump.At first device of the present invention is placed into by the fluorescent microscope.(camptothecine CPT) handled and was placed to after 12 hours in the container Jurkat cell culture (cell culture) with the camptothecine of 4x10-6mol/L.
CPT inducing cell generation apoptosis, this can be detected with Annexin V-FITC fluorescent dye.Phosphatidylserine (PS) is normally at the cell membrane inboard, but PS can be turned to surface of cell membrane from the cell membrane inboard in apoptotic cell.Green fluorescence dyestuff AnnexinV-FITC can combine with the PS specificity, therefore, is colored when Apoptosis and sends bright green fluorescence.Apoptotic cells then is not colored and does not show fluorescence.As shown in Figure 2, fluid loculus 12 is placed on the glove platform 4 of device of the present invention.By the speed of peristaltic pump the sampling of Jurkat cell culture is sent in the fluid loculus 12 with per minute 20 microlitres.Next can drive the displacement platform of a vertical direction to change glove platform (also being testing sample) and microscopic system 1 relative position (direction in vertical direction by knob 18 of manual rotation
), the distance between glove platform 4 and the microcobjective 3 in other words, thus can be so that sample accurately focuses on.Next computer control CCD camera obtains the cell suspension image that flows in the fluid loculus 12 with particular time interval (being 2 seconds in the present embodiment), thereby obtains a series of fluoroscopic images.Utilize the software of installing on computers that the image that is obtained is handled then.
The flow process of image signal processing program is almost identical with the embodiment that detects yeast activity, difference is, pixel value to G (green) passage in the pairing ellipse of cell is averaged, as the metric of judging this cell color, a threshold value is set in software program, and the cell that is higher than this threshold value is identified as apoptotic cell.Because apoptotic cell can not dyeed by Annexin V-FITC, therefore in fluoroscopic image, can not show.Therefore as if the ratio of wanting to learn that apoptotic cell is shared, also need fluorescent microscope is transformed into the bright field illumination mode to determine the quantity of total cell.
Detect cell green fluorescent protein (GFP) transfection efficiency
In the present embodiment, device of the present invention is applied to microscopic system microscope functions can be expanded to cell green fluorescent protein transfection efficiency detector.
The microscopic system of present embodiment is a fluorescent microscope, and signal processing unit is a computing machine, and image collecting device is a CCD camera, and the cell that is detected is the Chinese hamster ovary cell (CHO) of green fluorescent protein transfection after 24 hours.If the transfection success, sample itself can emitting fluorescence.
With reference to figure 1.At first device of the present invention is placed into by the fluorescent microscope.The cell DAPI fluorescent dyeing of sampling is placed on the carrier 5 then.Under microscope UV wave band excitation light irradiation, the cell of transfection green fluorescent protein success sends green fluorescence, and the cell of untransfected success sends blue-fluorescence.
Whole analysis process is similar to the embodiment that detects yeast activity, and the computing machine repetition is sent for 14 times and moved horizontally instruction, thereby obtains 15 width of cloth images of sample zones of different.Computing machine is handled the image that is obtained, automatically to blue, green cell is discerned and count.Program is averaged respectively to the pixel value of a pairing oval medium blue of cell (B) and G (green) passage, as the metric of judging this cell color.The corresponding judgment rule is set in software program, just can tells green and blue cell by right area, i.e. transfection successfully reaches not successful cell.The efficient of sample transfection can be calculated by following formula then:
Transfection efficiency=transfection success cell number/(the not successful cell number of transfection success cell number+transfection)
The analysis of cells growth cycle
Device of the present invention is applied to the analysis that the fluorescence microscopy system also can realize the fluidic cell ceremony of cell growth cycle.
The microscopic system of present embodiment is a fluorescent microscope, and signal processing unit is a computing machine, and image collecting device is a CCD camera.The embodiment of its operating process and detection yeast activity is (with reference to figure 1) much at one.At first the Chinese hamster ovary cell sampling is placed on the carrier 5.Cell has used the DAPI fluorescent dyeing in advance.Therefore DAPI is specific to combine with double-stranded DNA, and when using the light source irradiation of UV wave band, the cell that is in different growing stage is because of the dna content difference, thereby demonstrates different fluorescence intensities.The computing machine repetition is sent for 14 times and is moved horizontally instruction, thereby obtains 15 width of cloth images of sample zones of different.
Computing machine is handled automatically to the image that is obtained, the cell of various fluorescence intensities counted, and drafting statistic histogram as shown in Figure 9, drawing statistic histogram is a kind of common method of the attribute of cell being carried out graphical treatment.From this figure, can clearly offer an explanation out the distribution situation of cells in sample growth cycle.
The process step of image processing software is almost identical with the embodiment that detects yeast activity, and difference is that the B in the RGB passage (indigo plant) passage is averaged to the pixel value in the pairing ellipse of cell, as the metric of this cell fluorescence intensity.
Utilize bright-field microscope to detect cytoactive
Also common bright-field microscope can be expanded on function by device of the present invention is the active detector of automated cell.
The embodiment of operating process and detection yeast activity is (with reference to figure 1) much at one.At first the Chinese hamster ovary cell sampling is placed on the carrier 5.Cell uses common dye trypan blue (Trypan Blue) dyeing in advance, and wherein dying blue cell is dead cell, and the cell that is not colored is a living cells.The embodiment of its operating process and detection yeast activity much at one.The computing machine repetition is sent for 14 times and is moved horizontally instruction, thereby obtains 15 width of cloth images of sample zones of different.Computing machine is handled automatically to the image that is obtained, promptly to dyeing, the cell that is unstained discerns and counts, thereby learn the estimated value of the cytoactive in the sample.
The process step of image processing software is almost identical with the embodiment that detects yeast activity, difference is, pixel value to R (red) passage in the pairing ellipse of cell is averaged, metric as this cell dyeing degree, a threshold value is set in software program, the cell that is lower than this threshold value is identified as dead cell, otherwise then is considered as living cells.
T, bone-marrow-derived lymphocyte analysis in the blood
In the present embodiment, device of the present invention is applied to the detection that microscopic system can realize the fluidic cell ceremony, T, bone-marrow-derived lymphocyte in the blood are detected.
The embodiment of operating process and detection yeast activity is (with reference to figure 1) much at one.With erythrocyte cracked liquid the hematoclasis in the blood sample is fallen earlier before measuring.Add CD3-FITC and CD19-PE fluorescence antibody in the sample after the eccentric cleaning.The CD3-FITC antibody specificity with the T lymphocyte, CD19-PE antibody combines with bone-marrow-derived lymphocyte.Sample after handling is placed on the carrier 5, carrier 5 is placed on the glove platform 4 of apparatus of the present invention then.Under microscope blue wave band excitation light irradiation, the T lymphocyte will send green fluorescence, and bone-marrow-derived lymphocyte sends orange colour fluorescence.
The computing machine repetition is sent for 24 times and is moved horizontally instruction, thereby obtains 25 width of cloth images of sample zones of different.Computing machine is handled automatically to the image that is obtained, and promptly green, orange cell is discerned and is counted, thereby learn the estimated value of the cytoactive in the sample.
The process step of image processing software is almost identical with the embodiment that detects yeast activity, and difference is that program is averaged respectively to the pixel value of R (red) in the pairing ellipse of cell and G (green) passage, as the metric of judging this cell color.The corresponding judgment criterion is set in program, just can right area tells green and bisque cell, i.e. T lymphocyte and bone-marrow-derived lymphocyte, and count respectively.
Though the present invention discloses as above with preferred embodiment; right its is not in order to qualification the present invention, any those skilled in the art, without departing from the spirit and scope of the present invention; when can doing a little modification and perfect, so protection scope of the present invention is when with being as the criterion that claims were defined.
Claims (15)
1. servicing unit that is used for microscopic system, this servicing unit is independent of this microscopic system and comprises:
Place the glove platform of sample, this glove platform be can't help the body load-bearing of this microscopic system;
The displacement governor motion connects this glove platform, is used to regulate sample on this glove platform and the glove platform and the relative position between this microscopic system; And
Signal processing unit is electrically connected this displacement governor motion, is used to control regulating to small part of this displacement governor motion and moves, and control an image collecting device collected specimens image.
2. servicing unit as claimed in claim 1 is characterized in that, this servicing unit and microscopic system are without any fixedlying connected.
3. servicing unit as claimed in claim 1 is characterized in that, also comprises a liquid acquisition device, in order to the fluid supply collection liquid sample outside this glove platform.
4. servicing unit as claimed in claim 3 is characterized in that, this liquid acquisition device comprises:
The fluid loculus is positioned on this glove platform; And
Pump, in order to send into this fluid loculus from this fluid supply to this fluid loculus, this pump is connected to this signal processing unit.
5. as any described servicing unit of claim 1-3, it is characterized in that, also comprise at least one this image collecting device.
6. as any described servicing unit of claim 1-3, it is characterized in that, also comprise at least one wave filter, be used to handle light from sample.
7. as any described servicing unit of claim 1-3, it is characterized in that, also comprise detector, be used to measure the attribute of the illumination light of microscopic system.
8. as any described servicing unit of claim 1-3, it is characterized in that, also comprise at least one light source.
9. householder method that is used for microscopic system of using the described servicing unit of claim 1 may further comprise the steps:
This servicing unit is placed into by the microscopic system;
Sample is imported on the glove platform of this servicing unit;
Use this displacement governor motion to regulate relative position between this glove platform and this microscopic system; And
Use this signal processing unit to control an image collecting device collected specimens image.
10. householder method as claimed in claim 9 is characterized in that, also comprises sample is sought Jiao automatically.
11. householder method as claimed in claim 10 is characterized in that, this is sought Jiao automatically and comprises:
Sample is progressively moved in vertical direction, and to the sample imaging;
A reference value of calculation sample image;
Pairing with the highest reference value in a plurality of reference values as distinct image.
12. as claim 9 or 10 described householder methods, it is characterized in that, also comprise sample is carried out a kind of among the following analysis: cytoactive analysis, Apoptosis analysis, the analysis of cell green fluorescent protein transfection efficiency, cell growth cycle analysis, lymphocyte analysis.
13. as claim 9 or 10 described householder methods, it is characterized in that, comprise the algorithm of oval detection of image utilization or circle detection is handled.
14., it is characterized in that, use this displacement governor motion that the relative position between sample and the microscopic system is carried out coarse adjustment and/or fine setting, and coarse adjustment and/or the initial position of fine setting before each the measurement remain unchanged as claim 9 or 10 described householder methods.
15., it is characterized in that as claim 9 or 10 described householder methods, comprise that also the particle in this sample is analyzed, wherein this sample is at least one in organizing with next: medium, suspension, cell, cell culture, tissue, blood, body fluid; This particle is at least one in organizing with next: cell, biomarker (biomarker), protein, DNA, RNA.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE201020006598 DE202010006598U1 (en) | 2010-05-11 | 2010-05-11 | Device for microscopy |
| DE202010006598.4 | 2010-05-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102289065A true CN102289065A (en) | 2011-12-21 |
Family
ID=42675434
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011201619218U Expired - Lifetime CN202083829U (en) | 2010-05-11 | 2011-05-11 | Auxiliary device for microscope system |
| CN201110128927XA Pending CN102289065A (en) | 2010-05-11 | 2011-05-11 | Auxiliary device and method for microscope system |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011201619218U Expired - Lifetime CN202083829U (en) | 2010-05-11 | 2011-05-11 | Auxiliary device for microscope system |
Country Status (2)
| Country | Link |
|---|---|
| CN (2) | CN202083829U (en) |
| DE (1) | DE202010006598U1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111272724A (en) * | 2020-04-02 | 2020-06-12 | 深圳华因康基因科技有限公司 | Fluorescence detection optical system |
| CN113791095A (en) * | 2021-11-18 | 2021-12-14 | 煤炭科学研究总院 | Accurate sample position adjusting method for CT scanning |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE202010006598U1 (en) * | 2010-05-11 | 2010-09-02 | Wei, Ning | Device for microscopy |
| CN104214607B (en) * | 2014-08-28 | 2016-04-27 | 江阴新基电子设备有限公司 | Multichannel multi-angle combined light source device in vision detection system |
| CN106525865A (en) * | 2016-11-30 | 2017-03-22 | 南京理工大学 | Wafer image analysis device and method based on image processing |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293377A (en) * | 1999-10-04 | 2001-05-02 | 莱卡显微系统韦茨拉尔股份有限公司 | Device used for changing objective lens of microscope |
| JP2004198996A (en) * | 2002-12-20 | 2004-07-15 | Nikon Corp | Erect image microscope |
| CN101339185A (en) * | 2008-06-23 | 2009-01-07 | 武汉呵尔医疗科技发展有限公司 | Automatic microscopic imager for detecting cast-off cells and detection method |
| CN202083829U (en) * | 2010-05-11 | 2011-12-21 | 韦宁 | Auxiliary device for microscope system |
-
2010
- 2010-05-11 DE DE201020006598 patent/DE202010006598U1/en not_active Expired - Lifetime
-
2011
- 2011-05-11 CN CN2011201619218U patent/CN202083829U/en not_active Expired - Lifetime
- 2011-05-11 CN CN201110128927XA patent/CN102289065A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293377A (en) * | 1999-10-04 | 2001-05-02 | 莱卡显微系统韦茨拉尔股份有限公司 | Device used for changing objective lens of microscope |
| JP2004198996A (en) * | 2002-12-20 | 2004-07-15 | Nikon Corp | Erect image microscope |
| CN101339185A (en) * | 2008-06-23 | 2009-01-07 | 武汉呵尔医疗科技发展有限公司 | Automatic microscopic imager for detecting cast-off cells and detection method |
| CN202083829U (en) * | 2010-05-11 | 2011-12-21 | 韦宁 | Auxiliary device for microscope system |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111272724A (en) * | 2020-04-02 | 2020-06-12 | 深圳华因康基因科技有限公司 | Fluorescence detection optical system |
| CN111272724B (en) * | 2020-04-02 | 2023-09-05 | 深圳华因康基因科技有限公司 | Fluorescence detection optical system |
| CN113791095A (en) * | 2021-11-18 | 2021-12-14 | 煤炭科学研究总院 | Accurate sample position adjusting method for CT scanning |
| CN113791095B (en) * | 2021-11-18 | 2022-02-11 | 煤炭科学研究总院 | Accurate sample position adjusting method for CT scanning |
Also Published As
| Publication number | Publication date |
|---|---|
| DE202010006598U1 (en) | 2010-09-02 |
| CN202083829U (en) | 2011-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN202083829U (en) | Auxiliary device for microscope system | |
| US10578469B2 (en) | Automated set-up for cell sorting | |
| US10234392B2 (en) | Optical engine for flow cytometer, flow cytometer system and methods of use | |
| JP7129909B2 (en) | Systems and methods for characterizing particles in fluid samples | |
| US20050051723A1 (en) | Examination systems for biological samples | |
| CN110573858B (en) | Apparatus, system and method for imaging flow cytometry | |
| JP2009204616A5 (en) | ||
| JP2009204616A (en) | Apparatus for reading signals generated from resonance light scattering particle labels | |
| JP2011510292A (en) | Method and apparatus for analyzing particles in a liquid sample | |
| JP6513802B2 (en) | Laser light coupling for nanoparticle detection | |
| WO2020007326A1 (en) | High-throughput parallel raman spectrometer based on single cell detection | |
| CN102707425B (en) | Image processing method and device | |
| US8633432B2 (en) | Reflective focusing and transmissive projection device | |
| CN210015041U (en) | Microscopic imaging system | |
| US11371929B2 (en) | Systems, devices and methods for three-dimensional imaging of moving particles | |
| CN102224407A (en) | Method and apparatus for rapid filter analysis of fluid samples | |
| CN113155814A (en) | Portable colorimetric array image acquisition device based on optical fiber array and detection method | |
| WO2021148465A1 (en) | Method for outputting a focused image through a microscope | |
| CN209542943U (en) | A kind of cage construction laser scanning co-focusing micro imaging system | |
| CN110888230A (en) | Intelligent biological microscope | |
| CN111337416A (en) | Multi-channel sheath flow structure and label-free micro-fluidic cytometer and method thereof | |
| US20230360413A1 (en) | Methods for distinguishing particles in a fluid sample | |
| Han et al. | Imaging flow cytometry using linear array spot excitation | |
| KR102390074B1 (en) | The Apparatus for Continuously Monitoring Image of Microalgae | |
| CN202710835U (en) | Image processing device |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20111221 |