CN101310173A - Apparatus and method for detecting activity of living cells - Google Patents
Apparatus and method for detecting activity of living cells Download PDFInfo
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- CN101310173A CN101310173A CNA2006800412734A CN200680041273A CN101310173A CN 101310173 A CN101310173 A CN 101310173A CN A2006800412734 A CNA2006800412734 A CN A2006800412734A CN 200680041273 A CN200680041273 A CN 200680041273A CN 101310173 A CN101310173 A CN 101310173A
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Abstract
An apparatus for detecting the activity of living cells comprises a cell growth medium layer (104) for receiving the cells (100) and an imaging sensor (101) , e.g. a CCD array, wherein the cell growth medium layer and the imaging sensor are separated from each other only by an inert protective layer (102) and an optional diamond-like carbon layer (111) . The cells may be stained by a fluorescent dye, such as a calcium, ion, voltage or pH indicator dye. Upon illumination by a light source (130) the cells emit fluorescent light which is directly detected by the imaging senor without any optical transfer elements such as lenses or mirrors. The apparatus ,may be enclosed in a housing (103) provided with gas and liquid ports (107, 108, 109) . Furthermore the apparatus may comprise means (120) for applying a stimulation signal to the cells.
Description
Present patent application requires the right of priority at the u.s. patent application serial number 60/722,680 of submission on September 30th, 2005, and by reference it is combined in this, just as set forth it comprehensively.
Technical field
The present invention relates generally to cell detection, and more specifically, relate to the fluoroscopic examination of the neuronal activity that uses imaging sensor, and relate to the optical detection of the cytoactive of swarm cell type.
Background technology
Cytology relates to the formation of cell, a biological branch of 26S Proteasome Structure and Function research.When using in laboratory environment, scientist or other medical professionals examine cells for example, are determined the function of cell or are carried out the medical diagnosis of patient's illness.
Observation of minute biological samples is for example undertaken by microscope on the typical case in observation ground of cultured cells.When the record micro-image, use the camera or the video camera that are installed on the microscope.In addition, if especially must keep the temperature and humidity of sample constant, then may need to install or be installed in miscellaneous equipment on the microscope position near microscope position.Such arrangement may be expensive and complicated.
Develop various neural electron devices and be used for monitoring simultaneously the electroactive of small amounts of cells.Usually, these have used the extracellular recording of the action potential with extracellular microcircuit electrod-array; Yet, many uses that relate to the iuntercellular microelectrode.Neuron is pierced through by the micron-size electrodes of penetration cell wall.By cell death that wound caused usually within the several hrs that electrode inserts.These methods are further limited in and they can not be expanded to very large array.
Disclosed neural electron device depends on the electrostatic coupling between cell and the detecting device of being in that is used to monitor the neural network activity in U.S. Patent Publication 2004/0219184; Whole disclosures with described patent are combined in this by reference thus.
In addition, at U.S. Patent number 4,401, in 755,4,985,353,5,462,856,5,919,646,6,631,331,6,770,449,6,778,724 and 6,913,877 general description various detections and method of operating and/or diagnostic tool; Whole disclosures with described patent are combined in this by reference thus.
Thereby, the purpose of this invention is to provide the device, the system and method that are used for effectively detecting cytoactive, and need not complicated and expensive optical element.
Summary of the invention
Usually, the present invention relates to be used for sensing location closely near device, the system and method for the neuronal activity of the living cells of imaging sensor.Pair cell is handled, and making them to respond stimulates and fluoresce or luminous.In one embodiment, the cell of use is a neuron; Yet other cell material expects, and within the scope of the present invention.The advantage that the present invention surmounts prior art is do not have optical lens between nutrient culture media of observing and detecting device.Also be not used in the catoptron in the path of adjusting light.In detector array and corresponding one by one by existing between the zone of this array sensing.
In one aspect, the present invention relates to be used to detect the system of cytoactive.This system comprises being used to receive and is arranged on the imaging detector or near the equipment of cell material.The equipment that is used to receive cell material can comprise and is used for protective seam and/or diamond-like carbon-coating that cell material and imaging detector are isolated.In various embodiments, imaging detector is the array of sensor or sensor region, and can comprise charge-coupled device (CCD), complementary metal-oxide-semiconductor device (CMOS), charge injection device (CD), video camera, diode array or similar device.This system can also comprise the processor that is used to collect and handle the data that produced by this system, be used to the equipment that stores described memory of data and be used to show these data.
Described system can also comprise light source, for example, and such as laser instrument, lamp (for example incandescent, halogen or fluorescence) or light emitting diode.Light source can provide the light that is in various wavelength or multi-wavelength.Light source can also comprise power supply; Wave filter, for example notch filter or dichroic filter; And for example be used for to focus on by the light beam that light source is supplied with or the lens of calibration.
In various embodiments, described system comprises the equipment that is used to operate and/or handle cell material.For example, described system can comprise the equipment that is used for introducing to the environment of cell material gas, or is used for being used for for example equipment of the test of toxin from the environmental sampling of cell material.
In one aspect of the method, the present invention relates to a kind of method that detects cytoactive.The light of the wavelength that this method comprises the following steps: to provide such, the light of described wavelength are suitable for the dual ionization calcium ion (Ca from neuron cell body for example
++) tracking dyestuff stimulation fluorescent emission; Collect at least a portion of emitted fluorescence; Neuron about the activation of sensor location; And the time (temporal) and/or the amplitude that characterize detection signal.
In one embodiment, the collection of the light of emission is to realize by the array that is located immediately at light-sensing site (pixel) below the cell in the near field, and without any need for the optical delivery assembly, for example lens or catoptron.
In various embodiments, can detect on the top that may be positioned at detecting device, or closely near the arbitrary cell process on sagging (lop) of the tray deck of the light of imaging detector.In addition, cell material can be mixed, or otherwise handle.For example, some neurons can be handled with a kind of fluorescent material of following the trail of calcium or potassium ion, or this material can be handled with the photon-emitting compound that is used for voltage-sensitive dye that ion follows the trail of, is used to follow the trail of cell plasma passage or the motion of living cells intermediate ion.
By following description of reference and accompanying drawing, these and other objects of the present invention disclosed herein will become obvious together with advantage and feature.In addition, should be understood that the feature of various embodiments described herein is not to repel mutually, but can exist with various combination and permutation.
The accompanying drawing summary
In the accompanying drawings, same reference marker typically refers to identical part in all different views.In addition, accompanying drawing needn't be proportional and emphasis generally is to be used to illustrate principle of the present invention.In the following description, by describing various embodiments of the present invention, wherein with reference to following accompanying drawing:
Fig. 1 is the schematic elevational view of device that is used to detect cytoactive according to one embodiment of the invention;
Fig. 2 is the schematically showing of total system that is used to detect cytoactive according to one embodiment of the invention;
Fig. 3 is a part of schematic elevational view of device that is used to detect cytoactive according to one embodiment of the invention; With
Fig. 4 is the process flow diagram of explanation according to the method for the detection cytoactive of one embodiment of the invention.
Detailed Description Of The Invention
Embodiment of the present invention are below described.Yet, know to be pointed out that the present invention is not restricted to these embodiments, but be intended to be, also comprise obvious for those skilled in the art variation, change and equivalent.For example, the typical case describes device disclosed herein, system and method by measuring neuronal activity with reference to the cell material that uses some dyestuff and fluorescence; Yet clear being pointed out that can be implemented the present invention with cell material, dyestuff and the light source of other type.
As shown in fig. 1, neuron (100) grown or be placed on the top of imaging detector or sensor (101), and without any lens between two parties or the optical relay system of catoptron, optical fiber or any other type.Be assembled into image-position sensor, make its injured neuron not in its whole operation.For example, can between neuron and imaging sensor, place inertia protective seam (102) and diamond-like carbon-coating (111).The example of inertia protective seam and diamond-like carbon-coating has description in U.S. Patent Publication 2004/0219184.
In cell culture atmosphere (106), growth neuron in the cell growth medium (104) in liquid cellular incubation nutrition bath (105).Described nutrition bath and atmosphere are (103) of encapsulation and pass through port (108) by the extraneous gas source by port (107) and outer liquid body source and supplied with.Can extraneous gas and fluid supply be delivered to suitable port by the pipeline or the similar transportation equipment of any necessity.This system also comprises cell liquid discharge outlet (109), cell atmosphere switch (110) and is used for introducing to cell material the equipment (120) of signal.
When action potential passes the cell body propagation, produce fluorescence.Some light will be detected by imaging sensor, show the existence of neuronal activity.Should be pointed out that technology described here used the optical detection of fluorescence emission, and never rely on electrostatic coupling.
Imaging detector is made up of the sensing site that can sensing photonic or the array of pixel.Such device can be CCD, CMOS CMOS active pixel sensor, or is suitable for any other device with sensing site array of visible or near infrared imaging.Because photon is closely near the imaging sensor emission, so they are propagated in the near field.
Fig. 2 has described the total system layout of a preferred embodiment, comprises image device and auxiliary element.Can will cultivate atmosphere and nutritional fluid in other gas (210) and liquid (220) mixes.Like this, described device can be used to test the different chemical medicine of toxin or liquid or gas to being in the influence of the cell under cultivating.Can will show reaction by the variation on the cell behavior of sensor array detection to the chemicals of introducing.This system includes the introducing of all gases that helps be used in this system and liquid, the valve and the pipe fitting of necessity of shifting out and monitoring.
Following description is the general description according to the operation of an embodiment of system of the present invention.In operation, use image device (that is, when needed, the afterimage of imager is removed, and imager is set with its exposure mode of collecting photon wherein) with standard mode; Cell with some mode irriates or they from-stimulate, thereby luminous; And the photon of collecting is converted into digital form.
In a specific embodiments, imager is the CCD that at first has been eliminated any stored charge.Should be integral mode with its clock wave setting, wherein photon be converted into photoelectron in pixel well (pixel well).Apply cytositimulation, the radiation of fluorescence-stimulation will be shone this device, and will expose.After one period, exposure will stop, and read CCD with standard mode.The image that obtains will show the bright area that fluorescence occurred.
Can obtain a series of images, and the difference between the various image will illustrate the variation on the cytoactive.This is similar to checks continuous photo to distinguish variation, a kind of in the medium-term and long-term technology of using of many different scientific domains.For example, early stage in 20th century, Henrietta Leavitt use this technology find near variable in the galaxy.By measuring the brightness of the fixed star change in brightness, and make their position and brightness change lists in time, she surpasses 1000 variables classification.
Various embodiments of the present invention comprise that use is from Invitrogen (Carlsbad, the ion indication fluorescent dye with fluoro-3 dye forms CA).This is that a kind of calcium ion is followed the trail of dyestuff, and it is by being attached to dual ionization calcium ion (Ca
++) antibody form.What those skilled in the art will appreciate that is, in the context of the present invention, can also use other ion, pH or the voltage indicating dye that works with identical general fashion.Can apply action potential stimulus signal (112) to neuron.Neuron with action potential when its cell body is transmitted to cynapse, Ca
++Ion stasis will be crossed in the zone of the cell body that is close to cynapse at action potential.By rayed cell culture medium with the suitable wavelength of the about 490nm of nominal, will be by Ca
++The light of emission of ions wavelength about 505 to about 535nm, the about 515nm of peak value.That is, the light of this 515nm will be indicated the neuronic position of having provided.Can the light of this 515nm and the light of 490nm be distinguished by the placement of optical filter, described optical filter blocks the following light of 500nm, and the above light of transmission 500nm.In this embodiment, this filter plate should be placed on imaging detector (101) (in this case, CCD) and between the protective seam (102).Should be from total distance at bottom to the top of imaging sensor of neurocyte less than about 1mm (referring to Fig. 3).Fig. 4 has illustrated said process in process flow diagram.
In an alternative embodiment that is similar to the embodiment of just having described, do not use optical filter.Instead, the detected light signal that improves a little from irritation cell will be indicated nerve signal where passing through in array in the direct pixel below cell.
In yet another embodiment of the present invention, the dyestuff of voltage-activation can replace fluorescent dye to use.The dyestuff of voltage-activation is in response to variation in the voltage and emission light.Voltage-sensitive dye can be injected in the cell, or bathe introducing by nutritional fluid.Cross over when being in the neuron propagation that detects down single when action potential, will launch light by dyestuff.To launch light similarly by the continuous neuron of activation.
In yet another embodiment of the present invention, can use cell type except that neuron.To handle these cells, and make them to fluoresce or launch other signal in response to stimulating or not stimulating.This device will allow the user (for example, scientist) of system to set up rapidly and the quantization ratio compound to the metabolic effect of the cell that is being studied.Operable cell comprises for example cardiac muscle cell, endothelial cell, epithelial cell and reproduction cell.
Another embodiment of the invention will be utilized for example muscle cell of swarm cell type.The dynamic motion of one or more cells will be used as the indicator of metabolic activity.The detection of cell movement can be undertaken by bright field, fluorescence or other cell irradiation mode.
In previous embodiments, CCD can be used for shrinkage ratio and the amplitude of the cardiac muscle cell on the top that sensing directly is placed on pixel (chip sensor).Can come the sensing toxic agent by the variation on contraction ratio and the amplitude (increase or reduce).Be used to induce the stimulation of core cell contraction to be, but be not limited to, stimulate the extracellular potassium of electrical field stimulation, chemical stimulation or the increase of combination separately or with other.The sensing of shrinking can realize by luminous on the CCD that puts together with core cell (bright field illumination).CCD will be as the camera that is used to write down the cellular contraction that stimulates-induce.Except that ccd sensor, also expection can be placed on other sensor on the chip, and for example nano wire maybe can be measured other voltage-sensitive device of the action potential that is produced by myocyte or other cell.
Above-mentioned light source can be the illuminator of bright field direct electromotive force arbitrarily.The advantage that this technology surmounts other technology is, does not need to use in order to resolve fluorescence or the electrostatic detection methods of toxin to the influence of ion channel.Because the present invention do not need fluorescence, therefore do not need fluorescence light source and be used for only seeing optical filter by the dyestuff emitted fluorescence.In addition, the elimination that needs for electrostatic detection has been removed for the demand to the responsive chip of nerve or muscle cell discharge (action potential).The present invention can use single myocyte or myocyte's sheet.Lacking and existing the contraction data that to analyze under two kinds of situations of toxin under specific frequency of stimulation, for example ratio and amplitude.
Described certain embodiments of the present invention, will be obvious that, under the situation that does not deviate from the spirit and scope of the present invention, can use other embodiment that combines notion disclosed herein for those of ordinary skill in the art.Described embodiment goes up all will be understood that it only is illustrative in all fields, and nonrestrictive.
Claims (20)
1. device that is used to detect activity of living cells, described device comprises solid support, described solid support has:
A) cell growth medium layer;
B) inertia protective seam;
C) light source; With
D) detecting device,
E) be used to collect and handle the processor of the data that produce by described system;
F) be used to store the memory device of described data; With
G) be used to show the display device of described data,
Wherein said activity is detected by described device, and optical delivery assembly that need not be any for example lens or catoptron.
2. device according to claim 1, wherein said support is packed, and can comprise the liquid growth medium.
3. device according to claim 2, wherein said support have at least one inlet/outlet port.
4. device according to claim 1, described device also comprise insert described cell growth medium layer a) and described inertia protective seam b) between layer d of diamond-like-carbon).
5. device according to claim 1, wherein said detecting device can detect light emission or transmission, change in voltage and cell movement.
6. device according to claim 5, wherein said detecting device is selected from the group of being made up of following: charge-coupled device (CCD), complementary mos device (CMOS), charge injection device (CD) and diode array, or similar solid-state photon detection means.
7. device that is used to detect activity of living cells, described device comprises: imaging sensor (101), inertia protective seam (102), package support (103), cell growth medium (104), liquid cell culture nutrition are bathed (105), cell culture atmosphere (106), first port (107) and second port (108), cell liquid discharge outlet (109), cell atmosphere switch (110), light source (130) and are used for equipment (120) to cell material introducing signal.
8. device according to claim 7, described device also comprise the diamond-like carbon-coating (111) that inserts between described cell growth medium layer (104) and the described inertia protective seam (102).
9. device according to claim 7, described device also comprise the optical filter (300) that inserts between described imaging sensor (101) and the cell (100) to be detected.
10. device according to claim 7, described device also comprises first gas ports (205) that is connected to gas mixing valve (210), described gas mixing valve (210) is also connected to second gas ports (200), described gas mixing valve outlet is connected to gas access valve (215) and is connected to gas access port (107) then, described device also has the gas vent port (110) that is connected to outlet valve (220) that is communicated to device (103) outside, in addition, described device comprises first fluid port (230) that is connected to liquid mixing valve (235), liquid mixing valve (235) is also connected to second fluid port (225), described liquid mixing valve outlet port is connected to liquid entrance valve (240) and is connected to liquid inlet port (108) then, and described device also has the liquid outlet port (109) that is connected to liquid outlet valve (245) that is communicated to device (103) outside.
11. device according to claim 7, described device also comprises first gas ports that is communicated to device (103) outside, described first gas ports is connected to fluid intake port (109) by filter, described device also has the gas vent port (107) that is communicated to device (103) outside, and described device also has liquid outlet port (108).
12. one kind is used for detecting the active method of living cells, described method comprises the following steps:
A) cell of paying close attention to is put in the device of claim 1;
B) allow the cell absorption fluorescence indicator dyestuff of described concern to reach adequate time;
C) cellular exposure that makes described concern is in the light source that is in corresponding to the wavelength of the excitation wavelength of described indicator dye;
D) detect the fluorescigenic light emission be in corresponding to the wavelength of the emission wavelength of described indicator dye; With
E) amount of the light of analysis emission.
13. method according to claim 12, wherein said indicator dye are fluoro-3 or fluoro-4, and described excitation wavelength is between 490nm and 510nm, and described emission wavelength is between about 505nm and 535nm, preferably about 515nm.
14. method according to claim 12, wherein said indicator dye are the calcon-carboxylic acid dyestuff.
15. method according to claim 14, wherein said calcon-carboxylic acid dyestuff are selected from by fluorine derivative (fluo derivative) dyestuff, furan derivatives (fura derivative) dyestuff, indole derivatives (indo derivative) dyestuff, rhod-2, calcium is green and quin-2 forms group.
16. method according to claim 12, wherein said indicator dye are the ion indicator dyes.
17. method according to claim 12, wherein said indicator dye are voltage or pH indicator dye.
18. method according to claim 12, the cell of wherein said concern is a neuronal cell.
19. method according to claim 12, the cell right and wrong-neuronal cell of wherein said concern.
20. one kind is used for detecting the active method of living cells, described method comprises the following steps:
A) cell of paying close attention to is put in the device of claim 1;
B) make the cellular exposure of concern in light source;
C) take the image of the cell of described concern in particular time interval, and described image storage is used for subsequently analysis; With
D) difference in the described image of analysis.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US72268005P | 2005-09-30 | 2005-09-30 | |
| US60/722,680 | 2005-09-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101310173A true CN101310173A (en) | 2008-11-19 |
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Family Applications (1)
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| CNA2006800412734A Pending CN101310173A (en) | 2005-09-30 | 2006-09-29 | Apparatus and method for detecting activity of living cells |
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| EP (1) | EP1941262A1 (en) |
| JP (1) | JP2009509543A (en) |
| KR (1) | KR20080059593A (en) |
| CN (1) | CN101310173A (en) |
| AU (1) | AU2006297164A1 (en) |
| CA (1) | CA2624475A1 (en) |
| WO (1) | WO2007041308A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102549417A (en) * | 2009-09-30 | 2012-07-04 | 西门子公司 | Arrangement and method using microsensors for measuring cell vitalities |
| CN104321421A (en) * | 2012-03-30 | 2015-01-28 | 高丽大学校产学协力团 | Apparatus for measuring cell activity and method for analyzing cell activity |
| CN110132914A (en) * | 2019-04-28 | 2019-08-16 | 西南医科大学 | A method for optical imaging of myocardial slices in vivo |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2278332A1 (en) | 2009-07-20 | 2011-01-26 | Fondazione Centro San Raffaele del Monte Tabor | Method for optical measuring variations of cell membrane conductance |
| EP2870460A1 (en) * | 2012-07-03 | 2015-05-13 | Danmarks Tekniske Universitet (DTU) | An add-on system including a micro-reactor for an atr-ir spectrometer |
| KR101404607B1 (en) * | 2012-11-16 | 2014-06-11 | (주)실리콘화일 | On chip cell culture-monitor combine unit and combine kit using the unit |
| KR101505874B1 (en) * | 2012-12-05 | 2015-03-25 | (주)옵토레인 | On chip cell culture-monitor unit and combine kit using the unit |
| CN111065726A (en) * | 2017-09-07 | 2020-04-24 | 康宁股份有限公司 | Optical system for cell culture monitoring and analyte measurement |
| CN111103272B (en) * | 2018-10-26 | 2022-08-05 | 山东大学 | Real-time screening and measuring system and method for cell specific photosensitive effect |
| KR102562433B1 (en) * | 2020-09-04 | 2023-08-02 | 부산대학교 산학협력단 | Cell behavior analysis module, method for manufacturing cell behavior analysis module, and cell behavior analysis system comprising the same |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040219184A1 (en) * | 2003-03-25 | 2004-11-04 | The Regents Of The University Of California | Growth of large patterned arrays of neurons on CCD chips using plasma deposition methods |
-
2006
- 2006-09-29 CA CA002624475A patent/CA2624475A1/en not_active Abandoned
- 2006-09-29 KR KR1020087009871A patent/KR20080059593A/en not_active Withdrawn
- 2006-09-29 WO PCT/US2006/038093 patent/WO2007041308A1/en active Application Filing
- 2006-09-29 EP EP06825259A patent/EP1941262A1/en not_active Withdrawn
- 2006-09-29 CN CNA2006800412734A patent/CN101310173A/en active Pending
- 2006-09-29 JP JP2008533666A patent/JP2009509543A/en active Pending
- 2006-09-29 AU AU2006297164A patent/AU2006297164A1/en not_active Abandoned
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102549417A (en) * | 2009-09-30 | 2012-07-04 | 西门子公司 | Arrangement and method using microsensors for measuring cell vitalities |
| CN102549417B (en) * | 2009-09-30 | 2014-07-30 | 贝林格尔·英格海姆维特梅迪卡有限公司 | Arrangement and method using microsensors for measuring cell vitalities |
| CN104321421A (en) * | 2012-03-30 | 2015-01-28 | 高丽大学校产学协力团 | Apparatus for measuring cell activity and method for analyzing cell activity |
| CN104321421B (en) * | 2012-03-30 | 2016-06-01 | 高丽大学校产学协力团 | Cytoactive detection device and cell activation assay method |
| CN110132914A (en) * | 2019-04-28 | 2019-08-16 | 西南医科大学 | A method for optical imaging of myocardial slices in vivo |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2009509543A (en) | 2009-03-12 |
| CA2624475A1 (en) | 2007-04-12 |
| KR20080059593A (en) | 2008-06-30 |
| EP1941262A1 (en) | 2008-07-09 |
| AU2006297164A1 (en) | 2007-04-12 |
| WO2007041308A1 (en) | 2007-04-12 |
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