KR20080059593A - Apparatus and method for detecting activity of living cells - Google Patents

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

Apparatus and Method for Detecting Activity of Living Cells}

The present invention generally relates to the detection of cells, and more particularly to the fluorescence detection of neuronal activity and the optical detection of cellular activity of dynamic cells with an imaging sensor.

Background of the Invention

Cytology is the branch of biology that deals with the study of cell formation, structure and function. As applied in laboratory settings, scientists and other medical professors, for example, detect the function of cells and observe the cells for medical diagnosis of the patient's condition.

Observation of microbial samples, such as cultured cells, is typically done under a microscope. When a microscopic image is recorded, a camera or video camera mounted on the microscope is used. In addition, other devices are needed, especially if the temperature and humidity of the sample must be constant, mounted near or on the station of the microscope. Such an arrangement can be costly and complex.

In order to detect the electrical activity of a small number of cells, various neuroelectronic devices have been developed. In general, they use a method of extracellularly recording an action potential having an array of extracellular microcircuit electrodes. However, in some cases, intracellular microelectrodes are used. Neurons are penetrated by micron-size electrodes penetrating the cell wall. Within a few hours after electrode injection, apoptosis due to trauma is caused. These methods are extremely limited to avoid very large arrangements.

Neuroelectronic devices are disclosed in US Patent Publication No. 2004/0219184, which is incorporated by reference in its entirety. The device relies on a detector that can monitor the electrostatic coupling between cells and neural network activity.

In addition, various detection and processing methods and / or diagnostic tools are described in US Pat. No. 4,401,755; No. 4,985,353; No. 5,462,856; 5,919,646; 5,919,646; No. 6,631,331; 6,770,449; 6,770,449; 6,778,724; 6,778,724; And 6,913,877, the entirety of which is incorporated herein by reference.

Accordingly, it is an object of the present invention to provide an apparatus, system and method which can effectively detect the activity of a cell without the need for complicated and expensive optical components.

Summary of the Invention

The present invention relates generally to devices, systems, and methods for detecting activity of living cells located proximate to an image sensor. Cells are treated to fluoresce or emit light in response to a stimulus. According to one embodiment of the invention, the cell may be a neuron; However, other cellular components are also contemplated, and these are also within the scope of the present invention. One advantage of the present invention over the prior art is that there is no need for an optical lens between the medium to be observed and the detector. There is no mirror to adjust the light path. There is a one-to-one correspondence between the detector array and the area detected by the array.

According to another embodiment, the present invention relates to a system for detecting the activity of a cell. The system includes means for receiving cellular material placed on or adjacent to the image detector. The means for containing the cellular component comprises a diamond-like carbon layer for separating the cellular component from a protective layer and / or an imaging detector. In various embodiments of the present invention, an image detector is a sensor or an array of sensor areas and includes a charge-coupled device (CCD), a complementary metal-oxide semiconductor device (CMOS) ), A charge injection device (CD), a video camera, and a diode array or similar device. The system also includes a processor for collecting and processing data generated by the system, a storage for storing the data and a means for displaying the data.

The system includes, for example, a light source such as a laser, a lamp (eg, incandescent, halogen, or fluorescent), or a light emitting diode. The light source provides light at various wavelengths or multiple wavelengths. The light source is a power source; For example, a filter such as a notch or dichroic filter; And, for example, a lens for focusing or aiming at a beam of light provided from the light source.

In various embodiments of the present invention, the system comprises means for handling and / or treating cellular components. For example, the system includes means for introducing a gas into the cellular component environment or means for collecting the cellular component environment, for example for testing toxins.

According to another embodiment, the present invention relates to a method for detecting the activity of a cell. This is for instance double divalent calcium ions present in the nerve cell body (Ca ^{+ 2)} providing a light of an appropriate wavelength to stimulate fluorescence emission from the tracking dyes; Collecting at least a portion of the emitted fluorescence; Determining the location of activated neurons with respect to the sensor array; And analyzing the time and / or magnitude of the detected signal.

According to another embodiment of the invention, the collection of emitted light is a light-sensing site located directly below the cell in the near-field without an optical transfer assembly such as a lens or mirror. , Pixels).

In various embodiments of the invention, the activity of cells on the top of the detector or on the lop of a clear support plate proximate the image detector can be detected. In addition, the cellular component may be added with other substances or otherwise treated. For example, some neurons can be treated with phosphors that track calcium or potassium ions, or can be used with ion tracking, photon-emitting compounds that follow the movement of ions in cell ion channels or living cells. Can be treated with a voltage-sensitive dye.

In addition to the advantages and features of the present invention described herein, these and other objects will become apparent from the following description and the accompanying drawings. Moreover, the features of the various embodiments described herein are mutually exclusive and may exist as various combinations and permutations.

In the drawings, like numerals generally refer to the same parts in different drawings. In addition, it is not necessary to represent the size in the drawings to explain the principles of the present invention. In the following disclosure, various forms of embodiments of the present invention have been described with reference to the following drawings.

1 shows a schematic front view of an apparatus for detecting cell activity according to one embodiment of the invention.

Figure 2 is a schematic diagram of the entire system for detecting cell activity according to an embodiment of the present invention.

3 shows a schematic front view of a portion of a device for detecting cell activity according to one embodiment of the invention.

4 is a flowchart illustrating a method of detecting cell activity according to an embodiment of the present invention.

While various preferred embodiments of the present invention have been described in detail, the present invention is not limited to these embodiments and includes modifications, variations, and equivalents that are apparent to those skilled in the art. For example, the devices, systems and methods disclosed herein have conventionally been described for the detection of neuronal activity of cellular components using specific dyes and fluorescence, but the invention is also practiced with other types of cellular components, dyes and light sources. Could be.

As shown in FIG. 1, the neuron 100 grows or is positioned over an image detector or sensor 101 without any interfering lenses or mirrors, optical fibers, or other forms of optical relay systems. The image sensor is constructed so that the neuron does not go bad due to its operation. For example, an inner protective layer 102 and a diode-positive carbon layer 111 may be located between the neuron and the image sensor. Examples of inner protective layers and diode-positive carbon layers are disclosed in US Patent Publication No. 2004/0219184.

Neurons grow under cell culture gas 106 in cell culture medium 104 in a liquid cellular incubation nutrient medium 105. The nutrient bath and gas are encapsulated (103) and supplied by an external source of gas through port 107 and an external source of liquid through port 108. External sources of gas and liquid are delivered through the required tubes or other similar vehicles. The system includes a liquid drain (109), a cellular atmosphere tap (110), and means 120 for introducing a signal into the cellular component.

As the action potentials pass through the cell body, fluorescence is produced. Part of the light is detected by the image sensor, indicating that there is neuronal activity. The technique disclosed herein optically detects fluorescence and in no case relies on electrostatic coupling.

The image detector is configured as a sensing site, or array of pixels, that detects photons. Such a device may be a CCD, a CMOS active pixel sensor, or a device having a sensing site suitable for visible or near infrared imaging. Since the photons emit in proximity to the image sensor, they transition in the near-field.

2 shows the layout of the overall system of the preferred embodiment including the image elements and auxiliary elements. Other gases 210 and liquids 220 may be mixed with the culture gas and the nutrient solution. In this way, the device can be used to assay the effects of toxins or other chemicals on cells in culture, whether liquid or gas. Changes in cell behavior detectable by the sensor array may indicate a response to the chemicals introduced. The system includes valves and plumbing required for the injection, removal and monitoring of the various gases and liquids used in the system.

The following description is a general description of the operation of one embodiment of a system according to the invention. In operation, the image element is used normally (ie, the image element is placed in an exposure mode where it removes residual images and, if necessary, collects photons); Cells are stimulated or self-stimulated to emit light; The collected photons are converted into digital form.

According to one embodiment of the invention, the image element is a CCD, wherein the accumulated charge is preferentially removed. Its clock waveform can be set in an integration mode, where photons are converted to photo-electrons in the pixel wells. Cell stimulation may be applied, and fluoresecence-stimulating radiation may illuminate the device and exposure occurs. After a certain time, the exposure is terminated and the CCD is read in normal mode. The resulting image shows a bright region where fluorescence occurs.

A series of images can be obtained, and the differences between the various images indicate changes in cellular activity. This is analogous to observing a series of photographs to distinguish changes, a technique that has been employed for many years in different scientific spheres. For example, the technique was used by Henrietta Leavitt to observe stars in nearby galaxies in the early 20th century. By measuring the luminosity of stars whose luminosity changes, and tabulating the position and luminosity over time. He could list more than 1000 changing stars.

Various embodiments of the present invention include the use of ions indicating fluorescent dyes sold by Invitrogen in the form of flow-3. It is a calcium ion tracer dye and consists of an antibody that attaches to ^divalent calcium ions (Ca ²⁺ ). Those skilled in the art will appreciate that dyes exhibiting other ions, pH or voltages that function in the same general manner may be used in the present invention. Active potential stimulation signal 112 may be applied to neurons. When a neuron delivers an action potential to a synapse through a cell body, calcium ions in the cell body region where the action potential crosses adjacent synapses are collected. Mainly illuminated by giving light having a proper wavelength of about 490nm to the cell medium, the peak of about 515nm, about 505 to light of about 535nm wavelength to the Ca ^{2 +} ions Light emission. In other words, the position of the neuron is shiny 515nm light. An optical filter that excludes less than 500 nm but passes more than 500 nm can differentiate 515 nm light from 490 nm light. According to this embodiment, the filter is located between the image detector 101 (CCD in this case) and the protective layer 102. The total distance from the bottom of the neuron to the top of the image sensor is less than about 1 nm (see Figure 3). 4 shows a flowchart of the above-described process.

According to another embodiment similar to that described above, no light filter is used. Instead, a slightly upward light signal from the stimulated cell, detected in the pixel just below the cell, indicates where the array neural signal passes.

According to another embodiment of the present invention, a voltage-activated dye may be used instead of a fluorescent dye. The voltage-activated dye responds to a change in voltage and emits light. The voltage-sensitive dye may be injected into the cell or introduced through a nutrient solution bath. As the active potential passes through each individual nerve to be observed, light is emitted by the dye. Light is emitted by successive neurons that are similarly activated.

According to another embodiment of the invention, cell types other than neurons are used. These cells emit fluorescence or emit other signals in response to or without stimulation. The device quickly sets and quantifies the metabolic effects of compounds in the cells under study by the user of the system (eg, a scientist). Cells used include, for example, cardiomyocytes, endothelial cells, epidermal cells and regenerative cells.

Another embodiment of the invention utilizes dynamic cell morphology, such as muscle cells. The dynamic movement of the cell or cells is used as an indicator of metabolic activity. Detection of cell movement is accomplished through means of indicating bright field, fluorescence, or other cells.

According to the embodiment described above, the CCD can be used to detect the contraction rate and amplitude of the cardiomyocytes placed directly on top of the pixel (chip sensor). Toxic substances can be detected by changes (increase or decrease) in shrinkage and strength. Stimuli that induce cardiac cell contraction include, but are not limited to, electrical field stimulation, chemical stimulation, or increased extra-cellular potassium, or their interaction with other stimuli. May be a combination. Detection of contraction is achieved by shining light (bright field illumination) over the CCD on which the heart cells are placed. The CCD acts as a camera to record the stimulus-induced contraction of the cells. In addition to CCD sensors, sensors located on the chip, such as nanowires or other voltage sensing devices capable of measuring the active potential generated by muscle cells or other cells, can be considered.

The light source described above may be an irradiator driven by a bright field direct current. One advantage of the other prior art of the present invention is that it does not use fluorescence or electrostatic detection methods employed to solve the problem of the effects of toxins in ion channels. Since the present invention does not require fluorescence, it does not need a fluorescent light source and an optical filter for viewing the fluorescence emitted by the dye. Furthermore, no electrostatic detection requires a chip that is sensitive to nerve or muscle cell release (active potential). The present invention could use a single or sheet muscle cells. Contraction data, such as rate and intensity at certain frequencies of stimulation, can be analyzed in the presence and absence of toxins.

In the foregoing, specific embodiments of the present invention have been described, but those of ordinary skill in the art can use other embodiments including the concept described herein without departing from the spirit and scope of the present invention. I can understand that you can. It is to be understood that the above-described embodiments are merely illustrative and that the present invention is not limited thereto.

Claims (20)

(a) cell growth medium layer; (b) an inner protective layer; (c) a light source; (d) detectors; (e) a processor for collecting and processing data generated by the system; (f) storage means for storing data; And, (g) display means for displaying the data, A device for detecting the activity of living cells, the activity of which is detected by a device without an optical carrier such as a lens or a mirror. The method of claim 1, A support is provided and contains a liquid culture medium. Device. The method of claim 2, The support has at least one inlet / outlet port characterized in that Device. The method of claim 1, And a diamond-yang carbon layer (d) interposed between the cell growth medium layer (a) and the inner protective layer (b). Device. The method of claim 1, The detector can detect the emission or transmission of light, the change in voltage and the movement of the cell, characterized in that Device. The method of claim 5, The detector is a charge-coupled device (CCD), a complementary metal-oxide semiconductor device (CMOS), a charge injection device (CD), and a diode array or similar solid-state photons. Characterized in that it is selected from the group consisting of a solid solid state photonic detection device Device. Image sensor 101, inner protective layer 102, hermetic support 103, cell growth medium 104, liquid cell nutrient bath 105, cell culture gas 106, primary port ( 107 and a secondary port 108, a cell fluid drain 109, a cell atmosphere tap 110, a light source 130, and means 120 for introducing a signal into the cellular component Device for detecting the activity of living cells. The method of claim 7, wherein It further comprises a diamond-like carbon layer 111 interposed between the cell growth medium layer 104 and the inner protective layer 102 Device. The method of claim 7, wherein It further comprises an optical filter 300 interposed between the image sensor 101 and the cell 100 to be detected Device. The method of claim 7, wherein And further comprising a primary gas port 205 coupled to the gas mixing valve 210 that is also connected to the secondary gas port, and a gas mixing valve connected to the gas inlet valve 215 connected to the gas inlet port 107. In addition, it further comprises a gas outlet valve 110 connected to the outlet valve 220 in communication with the outside of the device 103, 1 coupled to the liquid mixing valve 235 connected to the secondary liquid port 225 And further comprising an outlet of the liquid mixing valve connected to the primary liquid port 230, the liquid inlet valve 240 connected to the liquid inlet port 108, and also in communication with the outside of the device 103. It characterized in that it comprises a liquid outlet port 109 connected to Device. The method of claim 7, wherein Further comprising a primary gas port in communication with the outside of the device 103 connected to the fluid inlet port 109 through the filter, having a gas outlet port 107 in communication with the outside of the device 103, Characterized in that it also has a liquid outlet port (108) Device. (a) positioning the cells of interest in the device described in claim 1; (b) allowing the cells of interest to absorb the fluorescent indicator dye for a sufficient time; (c) exposing the cells of interest to a light source having a wavelength corresponding to the excitation wavelength of the indicator dye; (d) detecting the emission of fluorescence having a wavelength corresponding to the emission wavelength of the indicator dye; And, (e) analyzing the amount of light emitted, How to detect the activity of living cells. The method of claim 12, The indicator dye is flow-3 (fluo-3) or flow-4 (fluo-4), the excitation wavelength is 490nm to 510nm, the emission wavelength is characterized in that 505nm to 535nm, preferably 515nm Way. The method of claim 12, The indicator dye is characterized in that the calcium indicator dye Way. The method of claim 14, The calcium indicator dye is a group consisting of flow derivative dye, fura derivative dye, indo derivative dye, Rhod-2, calcium green and quin-2. Characterized in that selected from Way. The method according to claim 12, The indicator dye is characterized in that the ion indicator dye Way. The method according to claim 12, The indicator dye is characterized in that the voltage or pH indicator dye Way. The method according to claim 12, The cell of interest is characterized in that the neuron (neuronal cell) Way. The method of claim 12, The cell of interest is characterized in that the non-neuron cells (non-neuronal cells) Way. (a) positioning the cells of interest in the device described in claim 1; (b) exposing the cells of interest to a light source; (c) taking an image of the cell of interest with a certain parallax and storing the image for later analysis; And, (d) analyzing the differences of the images, How to detect the activity of living cells.

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