WO2020083398A1 - 细胞特异性光敏效应的实时筛查与测量系统及方法 - Google Patents

细胞特异性光敏效应的实时筛查与测量系统及方法 Download PDF

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WO2020083398A1
WO2020083398A1 PCT/CN2019/113461 CN2019113461W WO2020083398A1 WO 2020083398 A1 WO2020083398 A1 WO 2020083398A1 CN 2019113461 W CN2019113461 W CN 2019113461W WO 2020083398 A1 WO2020083398 A1 WO 2020083398A1
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light
cells
fluorescence
ion
light source
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田岚
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Shandong University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells

Definitions

  • the invention belongs to the interdisciplinary fields of biology, medicine and optoelectronics, and particularly relates to a system and method for real-time screening and measurement of cell-specific photosensitization effects.
  • Biological cells contain various gated, ion-selective ion transport channels driven by solute concentration and transmembrane potential difference.
  • the activity of ion channels is regulated by channel protein on or off conformation in response to various stimulus signals (eg, light, electricity, heat, machinery, magnetism, etc.). Therefore, the ion channels on the cells can be stimulated by signals to achieve the function of regulating the signal transduction of nerve cells.
  • Ion channels are divided into light-sensitive ion channels, electric pressure channels, ligand gate channels, and pressure-activated channels according to the different protein response signals that constitute them.
  • Optogenetics is an emerging experimental technology that combines optics and genetics. It uses viral vectors to express light-sensitive proteins on regulated target cells or organ tissues, and uses specific parameters of light signals to regulate the glazing of cell membranes. The opening or closing of sensitive channels to precisely regulate the physiological function of cells, this technology has begun to be used in the treatment of certain neurological diseases.
  • the light-sensitive channel is a specific or selective ion channel with a transmembrane structure controlled by light pulses. It can quickly form a photocurrent and cause electrophysiological reactions in cells.
  • ChR2 Channelrhodopsin-2
  • the light-sensitive channel protein possesses an electrophysiological reaction that can rapidly form a photocurrent and depolarize cells.
  • Calcium is the second messenger in animal cells and participates in various functional activities of cells, such as contraction of muscle cells, secretion of glands, release of neurotransmitters, cell differentiation and neuronal death, etc. These important functional activities are accompanied by changes in intracellular calcium ion concentration.
  • calcium ions are usually not visible in the cells.
  • Calcium ion imaging technology is based on a strict correspondence between calcium ion concentration and nerve cell activity, using fluorescent dyes that calcium ions can bind (that is, calcium indicators). )), The changes in the concentration of calcium ions in neuronal cells are recorded by the changes in fluorescent signals, so that the signal transduction of nerve cells can be monitored.
  • the intracellular calcium ion concentration of a mammalian neuron at rest is 50-100nM, and the intracellular calcium ion concentration will increase significantly when the neuron is active.
  • HpHR Halhodopsin
  • ArchT ArchT
  • light-sensitive ion channel proteins are essential for the rapid excitation, rapid inhibition, and bistable regulation of nerve cells.
  • optogenetic technology transplants light-sensitive channel protein genes into corresponding biological cells through genetic technology, and expresses them to produce light-sensitive channel proteins, which is a research hotspot in biological regulation technology.
  • invention patents “Visualized Light Stimulation System and Visualized Light Stimulation Method” (CN200910132986.7) that use blue light (473nm) and yellow light (593nm) to realize photostimulation and imaging inspection of transgenic cells into which photosensitive proteins are introduced.
  • this patent uses only two bands of light for the stimulation light, and cannot detect the sensitive effects of more light bands of the cells to be tested.
  • the patent only conducts imaging experiments on transgenic cells treated with optogenetic technology to prove the effectiveness of the transgene, and it cannot screen any animal somatic cells for photosensitization effects, that is, its detection cell type is limited.
  • Some cells in nature may contain light-sensitive proteins or structures by themselves or after being modified by components, but these light-sensitive structures have a specific response to the light of which wavelength band, or whether a target cell has a light-sensitive effect, or if there is a light-sensitive effect, the corresponding specificity
  • the existing methods are limited to the expression verification of the structure or composition of the photosensitive effect, and do not have the ability to quickly find or screen.
  • the present invention provides an unlimited band (visible and non-visible) optical signal, no longer limited to transgenic cells, a universal and operable real-time screening system and method for cell-specific photosensitization effect, and can screen Quantitative and localization analysis of photosensitivity of cells.
  • the purpose of the present invention is to provide a method for real-time screening and measurement of cell-specific photosensitizing effects.
  • the method of the present invention is fast, repeatable, and can be widely used in biotechnology and life science and medical research in the field of optogenetics, and the The method can analyze the working state of photosensitivity from both quantitative and qualitative levels.
  • the invention can perform real-time screening on whether the cell to be tested contains a specific wavelength of photosensitization effect, and further measure the working condition of the photosensitization effect in real time; thereby confirming which wavelength bands the cells containing the photosensitization effect have a specific response to.
  • the cell to be tested may be a transgenic cell processed by optogenetic technology or an ordinary animal and plant cell that has not been processed by transgenic technology.
  • the wavelength of the stimulating light of the cell to be tested can be adjusted.
  • the stimulating light source to be tested can be a general-illumination type, or fiber-coupled output, or LED light emitting device and other forms of monochromatic light sources.
  • the stimulus optical output port can be added with beam shaping device or natural port output.
  • the light path with beam shaping facilitates better energy gathering, reduces system power consumption, and at the same time facilitates the precise positioning of the photosensitivity response of the cells to be tested.
  • the light path without beam shaping can increase the irradiated area of the stimulating light, increase the range of its photosensitivity response, and observe the response effects of the photosensitivity effects at different positions of the divergent light.
  • the present invention provides a method for real-time screening and measuring the photosensitivity of cells, the method comprising stimulating the cells to be tested with light to be tested A, and using cells B and / or C to stimulate the cells to generate light D, Fluorescence imaging of light D, based on the real-time change of ion concentration to determine the existence of photosensitization effect or further analyze its working condition;
  • the light A is a monochromatic pulse laser with various parameters such as wavelength (band), light intensity, pulse width, repetition rate, and irradiation duration (timing) that can be arbitrarily adjusted within a certain range to realize the screening of optical signals.
  • parameters such as wavelength (band), light intensity, pulse width, repetition rate, and irradiation duration (timing) that can be arbitrarily adjusted within a certain range to realize the screening of optical signals.
  • the cells to be tested are living cells in vitro.
  • various ions ions to be detected
  • calcium ions Ca 2+
  • an ion fluorescence indicator or fluorescent probe
  • calcium ion is the ion to be detected, and calcium ion fluorescent indicator or fluorescent probe is added.
  • the method detects the fluorescence intensity through ion imaging technology and processes the data, and then draws a specific curve of real-time or timed ion concentration changes to judge the existence of photosensitization effect or further analyze its working conditions.
  • the method of the present invention does not specify the type and type of cells. Therefore, the method of the present invention has Universality.
  • the light B and / or the light C are excitation light sources of the ion fluorescence indicator;
  • the stained cells that is, the cells to be tested with the ion fluorescence indicator added
  • the emitted fluorescence is light D;
  • the ion fluorescence indicator when the light B and the light C are excitation light sources of the ion fluorescence indicator, the ion fluorescence indicator has a characteristic excitation under the light B after combining with the free ion, and the light fluorescence under the light C when the ion fluorescent indicator does not combine with the free ion Feature excitation
  • the method includes using light A to stimulate the cells to be tested, and using light B and / or light C to perform fluorescence imaging on the cells, wherein when using light B and / or light C to perform fluorescence imaging on the cells, the fluorescence is detected
  • the intensity of D, imaging fluorescence D is detected
  • the method includes stimulating the fluorescently stained cells with the light to be tested A, and using the light B and / or light C to perform fluorescence excitation on the cells, and the fluorescence D generated by the excitation is further imaged by CCD fluorescence and transmitted to a computer Carry out data processing and drawing, analyze the real-time change of free ion concentration to determine the existence or response of photosensitive effect.
  • CCD fluorescence imaging add a filter set for light B and light C to eliminate the influence of excitation light B and light C on the final imaging result; or in the optical path of CCD fluorescence imaging, add only light D passes through the filter, so that only the light D is imaged.
  • the ion fluorescent indicator described in the present invention is a type of chemical substance with fluorescent characteristics for specific ions (such as calcium, potassium, sodium, etc.), which has many types and different chemical principles.
  • the absorption wavelength and emission wavelength of the ion fluorescence indicator are different, that is, the light B and or the light C are determined by the absorption wavelength, and the light D is the emitted fluorescence wavelength; some of the fluorescence indicator is composed of a single wavelength Excited by colored light, some fluorescent indicators are still excited by dual-wavelength monochromatic light. Table 1 summarizes the recommended wavelengths of light B and light C when using several common calcium fluorescent indicators.
  • Indicator name K d value Light B recommended wavelength Light C recommended wavelength Indo-1 230nM 405nm ------ Fura-2 140nM 340nm 380nm Fluo-3 400nM 490nm ------ Fluo-4 345nM 490nm ------ BTC 7mM 400nm 480nm Benzothiaza-1 660nM 340nm 380nm
  • the light B and / or light C can be determined according to the product instructions or guidelines.
  • the wavelengths of light A, light B, light C, and light D are different from each other; the center value of the band must be different, and the difference is at least tens of nm or more; the tens of nm is, for example, at least 10 nm, 15nm, 20nm, 25nm, 30nm, 40nm or 50nm and so on.
  • the light A is a single-band pulsed laser or a combination of light in a pulsed laser measurement sequence containing N adjustable parameters of n bands, where n and N are positive integers, n ⁇ 2, and N is The combination of the laser measurement order of n wave bands, generally N ⁇ n.
  • the light A when the light A is a combination of light of N pulse laser measurement sequences containing n bands, the light A is used to stimulate the cells to be tested. This measurement sequence stimulates the cells separately.
  • the N pulse lasers of the n bands can be freely switched; wherein, in some embodiments of the present invention, when the n pulse lasers of the n bands are freely switched to stimulate cells, respectively, the phase The frequency bands of pulsed laser light for two adjacent stimuli are different.
  • multi-band measurement multiple parameters of pulsed light can be adjusted arbitrarily, such parameters as light intensity, pulse width, repetition rate, timing, parameter adjustment is usually from small to large (or from weak to strong), n bands per group In the measurement, each parameter should be consistent to ensure the comparability of the measurement.
  • the center value of the band of the pulse laser of the two adjacent stimuli has a gap of 20 to 1000 nm, the gap is preferably 20 to 800 nm, more preferably 20 to 400 nm, and most preferably 40 nm.
  • the method includes the following steps:
  • Cell treatment Select the cells to be tested for in vitro culture, add calcium ions separately and / or rinse the culture dish with a solution containing calcium ions (such as HBSS solution) that can maintain cell activity for a short time; add calcium ion fluorescence indicator Reagent or fluorescent probe and incubate; select appropriate cells with fluorescent label under light B and light C;
  • a solution containing calcium ions such as HBSS solution
  • the method for culturing cells in vitro may use a method conventionally mastered by a person skilled in the art; or according to the following method for culturing cells in vitro: selecting cells and performing in vitro culture in a petri dish with cell slides, the temperature of the petri dish is controlled at At 37 °C, the gas environment is 95% air and 5% CO 2. Add a Petri dish suitable for cell culture (for example, if culturing nerve cells, you can choose DMEM-F12 medium specially used for nerve cell culture), and cultivate for 24 hours .
  • the incubation time can be selected according to the conventional method, or the incubation time is 30-60 minutes.
  • the calcium ion fluorescent indicator or fluorescent probe may be, for example, Fura-2, or other calcium ion fluorescent indicators as shown in Table 1.
  • Calcium ion fluorescence imaging the calcium ion fluorescence indicators are excited by light B and light C, respectively, to detect the fluorescence intensity; calculate the ratio of the fluorescence intensity F B / F C at the two excitation wavelengths of light B and light C , and calculate the free calcium ion concentration;
  • the concentration of the free calcium ion can be calculated according to the Grynkiewicz formula; the Grynkiewicz formula is expressed as follows:
  • K d is the equilibrium dissociation constant of the combination of a fluorescent indicator (such as Fura-2) and calcium ions, and its value is closely related to temperature, pH value, ion concentration, etc.
  • a fluorescent indicator such as Fura-2
  • Fura-2 is 224 at 37 °C
  • ⁇ is the ratio of fluorescence intensity of light C is zero calcium and saturated intracellular calcium
  • R is each measurement point F B / F C fluorescence intensity ratio
  • R min is zero calcium F B / F C fluorescence intensity ratio
  • R max is For saturated calcium, the F B / F C fluorescence intensity ratio, R max / R min value is between 13 and 25.
  • step (2) Fluorescence imaging of calcium ion under the stimulation of light A: On the basis of the method of step (2), first, the emission end of light A is fixed to ensure the distance between the light A and the sample and the angle of incidence. Then, the cells were stimulated with light A, and only the light D was passed by setting the filter group on the imaging optical path. During the light A stimulation, calcium ion fluorescence imaging was performed on the light D, and the changes were recorded. When the light A is in the invisible light band, it is necessary to use visible light to mark through the same optical path, fix the light A into the position, and replace the light A with the invisible light to be measured, and then perform fluorescence imaging. Finally, calculate the concentration of free calcium ions under light A stimulation.
  • step (2) Draw the free calcium ion concentration change curve according to the free calcium ion concentration obtained in step (2) and step (3) in real time or regularly. Compare the change of free calcium ion concentration without light A stimulation and light A stimulation on the ion concentration change curve to judge the existence of photosensitization effect or further analyze its working condition.
  • the free calcium ion concentration change curve can be drawn directly in step (2) or step (3) to achieve real-time synchronization. Finally, compare the change of free calcium ion concentration without light A stimulation and light A stimulation on the ion concentration change curve to judge the existence of photosensitization effect or further analyze its working condition.
  • the present invention detects the auditory nerve cells, including: using light A to stimulate the cultured auditory nerve cells in vitro (such as selecting spiral ganglion cells in the cochlear shaft), and the cells are in the environment Calcium ions are present and calcium ion fluorescent indicator is added. Cells B and C are used to perform fluorescence imaging on the cells. Based on the real-time change of free calcium ion concentration, the existence of photosensitization effect is judged or its working conditions are further analyzed. Specifically, the method includes:
  • Cell treatment Select auditory nerve cells (such as spiral ganglion cells) for in vitro culture, rinse the petri dish with HBSS solution (at least once); add calcium ion fluorescence indicator, such as Fura-2 and incubate for 30-60min; Appropriate cells with fluorescent labeling are selected under ultraviolet light at 340 nm and ultraviolet light at 380 nm; the appropriate means selecting cells in a fluorescently labeled cell imaging image with complete morphology and proper location distribution;
  • HBSS solution at least once
  • calcium ion fluorescence indicator such as Fura-2 and incubate for 30-60min
  • Appropriate cells with fluorescent labeling are selected under ultraviolet light at 340 nm and ultraviolet light at 380 nm; the appropriate means selecting cells in a fluorescently labeled cell imaging image with complete morphology and proper location distribution;
  • Calcium ion fluorescence imaging the calcium ion fluorescence indicator is excited by 340nm ultraviolet light or 380nm ultraviolet light, respectively, to detect the fluorescence intensity, calculate the ratio of fluorescence intensity F 340 / F 380 at two excitation wavelengths, and calculate the free Calcium ion concentration;
  • the free calcium ion concentration can be calculated according to the Grynkiewicz formula.
  • the Grynkiewicz formula is expressed as follows:
  • K d is the equilibrium dissociation constant of the combination of Fura-2 and calcium ions, and its value is closely related to temperature, pH value, ion concentration, etc., it is 224 at 37 °C; ⁇ is zero intracellular calcium and saturated calcium at 380nm Fluorescence intensity ratio; R is the fluorescence intensity ratio of F 340 / F 380 at each measuring point; R min is the fluorescence intensity ratio of F 340 / F 380 at zero calcium; R max is the fluorescence intensity ratio of F 340 / F 380 at saturated calcium, R The max / R min value is between 13 and 25.
  • Fluorescence imaging of calcium ions under the stimulation of light A On the basis of the method of step (2), cells are stimulated with light A, and fluorescence imaging of calcium ions is performed to calculate the concentration of free calcium ions under the stimulation of light A;
  • Light A is a pulsed laser, and its wavelength range can be, for example, any band or a combination of multiple bands from 450 nm to 1065 nm;
  • the wavelengths of the light from two adjacent stimulations have a wavelength difference of 20 to 1000 nm, and the wavelength difference is preferably 20 to 800 nm, more preferably 20 to 400 nm, or 40 nm; a more precise statement is ,
  • the wavelength difference is the difference in the center value of the band.
  • the light A when the light A is in the invisible light band, first use visible light to mark through the same light path or position, fix the light A into the position, replace the light A with the invisible light to be measured, and then perform fluorescence imaging.
  • step (3) Draw the free calcium ion concentration change curve according to the free calcium ion concentration obtained in step (2) and step (3) in real time or time; compare the free calcium ion stimulation curve with the free calcium under the light A stimulation on the ion concentration change curve The change in ion concentration determines the presence of photosensitizing effects on auditory nerve cells or further analyzes their working conditions.
  • Using the method of the present invention to explore the existence of the photosensitizing effect of the auditory nerve provides an important basis for studying the working mechanism of the auditory nerve under light stimulation, and has important significance for further research on the regulation of optogenetic technology in the auditory nerve and the application of clinical neurological diseases .
  • the present invention provides a system for real-time screening and measuring cell-specific photosensitizing effects, including:
  • Light source A which emits light A to stimulate the cells to be tested
  • An ion imaging system which includes at least a fluorescence excitation light path system and a CCD imaging system, the fluorescence collection light path excites different fluorescence, and a CCD imaging system detects the fluorescence intensity and performs image data collection;
  • a data processing system that processes data collected by the ion imaging system.
  • the light source A is a multi-band switchable, monochromatic pulse laser stimulation light source with adjustable parameters, and the parameters include light intensity, pulse width, repetition rate, and timing.
  • the light source A may emit visible light and non-visible light.
  • the light source A is coupled with the output light path 1, and the light A can be output in different periods by switching the light source A and / or adjusting the parameters of the light source A.
  • the light source A can output a single-band pulsed laser with adjustable parameters or can output pulsed lasers with n bands in N switching sequences, where n and N are positive integers and n ⁇ 2, N ⁇ n.
  • the light source A is a general-illumination light-emitting device, a fiber-coupled light-emitting device, or an LED light-emitting device.
  • the emission end of the light source A is an emission port with beam shaping or a natural emission port without beam shaping.
  • the position of the light source and the optical path is adjustable, and the position includes the distance between the remote port of the light source or the optical path and the measured point or the area to be measured, and the light A is directed to the point or area to be measured angle.
  • the adjustment of its position can be achieved by some kind of fixing device.
  • the fluorescent excitation light path system includes at least two light sources of B and C or at least a light source capable of switching or coexisting the two light sources of B and C;
  • the light source B is the characteristic excitation band light source when the fluorescent ion probe is combined with the corresponding ion, the characteristic excitation light is light B;
  • the light source C is the characteristic excitation band light source when the fluorescent ion probe is not combined with the corresponding ion, the characteristic excitation The light is light C;
  • the light source B and the light source C are coupled with the output optical path 2, and alternately output light B and light C to illuminate the cells to be tested and excite fluorescence D.
  • an observation optical path is provided in the CCD imaging system, and an optical sheet is provided on the observation optical path.
  • the optical sheet may be an optical sheet that can only transmit light D or may be simultaneously blocked.
  • Light sheet that cuts off light A, light B and light C to prevent the three kinds of light from transmitting through A; B and C;
  • CCD imaging system detects fluorescence through the observation light path with light sheet and collects image data, and the collected data is transmitted to the data Processing system.
  • the data collected by the CCD imaging system is transmitted to the data processing system through the data link, and the data processing system analyzes, calculates, and plots the data to obtain real-time data and images.
  • the system for real-time screening and measuring cell-specific photosensitizing effects of the present invention includes:
  • Light source A (that is, the light source to be screened), which is a multi-band switchable pulsed laser stimulation light source (the adjustment or free switching between the different wave bands can be achieved through, for example, a laser conversion interface or other components with this function Realization), light source A can adjust four parameters including light intensity, pulse width, repetition rate and timing;
  • the light source A is coupled to the output optical path 1 (such as an optical fiber, in the present invention, the optical fiber is also called the output optical fiber 1 and has a diameter of 100 ⁇ m) through a laser conversion interface (such as a flange converter), and the light source can be switched at different time periods Generate pulsed lasers of different wavelengths; the laser output from light source A is applied to the sample to be tested through output optical path 1 (such as output fiber 1, the fiber is not in contact with the sample); the light emitting device of light source A can be a fiber-coupled semiconductor laser or LED (such as ⁇ LED) and so on.
  • the emission end (also called output end) of the light source A is divided into two types, namely, an emission port with beam shaping and a natural emission port without beam shaping.
  • the light path with beam shaping facilitates better energy gathering, reduces system power consumption, and facilitates the precise positioning of the photosensitivity response of the cells to be tested, as well as reducing the influence caused by adjusting the position of the light path, and improving detection stability and sensitivity.
  • the light path without beam shaping can increase the irradiated area of the stimulating light, increase the range of its photosensitivity response, and observe the response effect of the photosensitivity effect of cells at different positions of the divergent light.
  • the sample to be tested is a cell cultured in vitro, and the ion to be detected exists in the environment where the cell is located and an ion fluorescent probe corresponding to the ion to be detected has been added;
  • the sample to be tested and the output end of the light A are fixed by a fixing device.
  • the operation table of the ion imaging system may have a small dish for holding the sample to be tested and a three-dimensional positioning bracket, which can fix the sample to be tested (such as a cell slide) And the output fiber; the fixing device can also fix the distance between the emitting end of the light A and the sample to be measured, and the selection of the incident angle of the light A; the output fiber and the sample to be measured must maintain a suitable distance, and must be ensured during the measurement The position of the cell slide and the output fiber are always fixed.
  • Ion imaging part which includes a fluorescent probe excitation light path system and a CCD imaging system
  • the light path includes at least two light sources B and C or can realize the switching and coexistence of the two light sources B and C, and the light source B and / or the light source C are characteristic excitation wavelength bands of the ion fluorescence indicator Light source; illuminate the sample to be tested by light source B or C, or alternately illuminate the sample to be tested by light source B and light source C (light is irradiated to the sample to be tested through output optical path 2, which is output fiber 2 for example), exciting different fluorescence Generate fluorescence intensity, the fluorescence generated by the excitation is fluorescence D;
  • CCD imaging system which detects the fluorescence intensity through observation optical path (such as CCD) and collects image data, and transmits the data to the data processing system through the data link;
  • observation optical path should include a wide-band adjustable band filter combination .
  • the filter can be a filter of light A, light B and light C, or a filter that can only transmit fluorescence D, thereby eliminating the interference of light A, B and C, and passing the collected fluorescence D CCD imaging;
  • observation optical path also includes an observation optical path branch leading to an optical microscope. Observe the cells in the field of view and the position of the output fiber through the observation eyepiece of the optical microscope. In order to ensure that the laser to be tested can be accurately irradiated to the cell to be tested through the output optical path 1 (output fiber 1).
  • the observation light path branch and the optical microscope connected to it can be removed or closed after determining the location of the cell and output fiber. It does not directly participate in the real-time screening and measurement of photosensitivity itself.
  • the data processing system may use, for example, MetaFluor fluorescence ratio imaging software, which can simultaneously display raw data, ratio images, fluorescence intensity graphs, ratio graphs, ion concentration graphs, and such as brightfield or Non-scale measurement images such as phase contrast imaging.
  • the software can simultaneously image and measure two different ratio determination indicators without being affected by dye loading concentration, conditions or emission intensity.
  • both the light output and input require an optical path (such as optical fiber, beam shaping, CCD), and the connection between the optical path and the corresponding device or component can be achieved by using a well-known circuit interface or conversion interface in the art.
  • optical path such as optical fiber, beam shaping, CCD
  • FIG. 2 the system of the present invention is shown in FIG. 2.
  • the present invention provides a method for screening and measuring cell photosensitivity in real time using the system shown above, the method comprising the following steps:
  • Cell treatment select the cells to be tested for in vitro culture, add the ions to be detected separately or rinse the culture dish with a solution containing the ions to be detected that can maintain cell activity for a short time; add the ion fluorescence indicator corresponding to the ions to be detected After the incubation, select appropriate cells with fluorescent labels under the excitation of light source B and / or light source C;
  • the cell treatment After the cell treatment is completed, remove the cell slide from the culture dish and place it in a small dish on the operating platform of the ion imaging system. Fix the position of the cell slide and the light A output port by adjusting the three-dimensional positioning bracket.
  • the cells should be kept at a suitable distance and not in contact with the cells; in order to avoid fluorescence quenching, the whole process should be carried out under dark light conditions. Observe the cells in the field of view and the position of the output A optical port through an optical microscope. Turn off the light of the optical microscope, and use the specific wavelength laser (excited by a fluorescent probe) corresponding to the specific fluorescent ion indicator to perform fluorescent labeling. In the fluorescently labeled cell imaging image, select cells with complete morphology and proper location distribution.
  • Ion fluorescence imaging the light source B and / or the light source C of the optical path system are excited by the fluorescent probe to excite the ion fluorescence indicator, and the free ion and the bound ion emitted by the fluorescent probe and the cell to be tested are respectively detected by the CCD imaging system
  • the fluorescence intensity resting value ie, fluorescence D, as described above
  • Ion fluorescence imaging under light source A stimulation turn on light source A to stimulate cells, repeat the operation of step (2), perform ion fluorescence imaging under light source A stimulation, and calculate the real-time or timing free ion concentration under light source A stimulation ;
  • the CCD imaging system detects the fluorescence intensity in steps (2) and (3) and transmits the data to the data processing system; the data processing system calculates the data and draws a real-time ion concentration curve;
  • the light source A only generates a single-band pulsed laser with specific parameters at the same time.
  • the light source A can switch the band at different times and adjust the parameters at any time.
  • the parameters include light intensity, pulse width, repetition rate, and timing;
  • the light source A can generate a combination of N pulse laser measurement sequences containing n bands, where n and N are positive integers, n ⁇ 2, N ⁇ n; N pulses of the n bands Freely switch between laser measurement sequences;
  • the wave bands of the pulse lasers of two adjacent stimulations are different;
  • the wavelength band of the pulse laser of the two adjacent stimuli has a gap of 20 to 1000 nm, the gap is preferably 20 to 800 nm, more preferably 20 to 400 nm, and most preferably 40 nm.
  • the wavelengths of light A, light B, light D, and light C are different from each other; wherein, in some embodiments of the present invention, the wavelengths of the light A, light B, light D, and light C (band center value )
  • the difference between them is at least tens of nm or more, such as 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm or 50nm, etc. (depending on the monochromaticity of the light source device).
  • the ion to be detected is calcium ion
  • the ion fluorescence indicator is selected from any one of Table 1.
  • FIG. 1 is a schematic diagram of the principle of the method of the present invention; wherein, (a) is the ion status inside and outside the cell that is not irradiated with light of a specific wavelength; (b) is the ion status inside and outside the cell that is irradiated with light of a specific wavelength.
  • FIG. 2 is a schematic diagram of a system for real-time screening and measuring cell-specific photosensitizing effects of the present invention.
  • the system contains three kinds of light sources, of which light source A (that is, the light source to be measured) is a pulse laser with parameters such as adjustable light intensity, pulse width, repetition rate, and timing to be screened.
  • the light source is a pulse laser with corresponding parameters emitted by an adjustable parameter laser or a light emitting device.
  • the generated pulse light signal with specific parameters is coupled to the output optical path 1 through the optical conversion interface and the light source is switched, and pulse light in different wave bands can be generated in different time periods.
  • the optical signal is irradiated to the corresponding sample cell to be tested through the output optical path 1.
  • the light source B and the light source C are two light sources in the excitation light path system of the fluorescent probe, which are characteristic excitation light sources when ions are combined with the probe and unbound.
  • the light source B and the light source C alternately irradiate the sample cell to be tested through the output light path 2 to excite the dyed sample to be tested to produce the corresponding fluorescence D, image data collection is performed by the CCD imaging system, and the data link is transmitted to data processing
  • the computer uses image processing software to calculate and draw data, and obtains the corresponding real-time ion concentration curve.
  • Figure 3 shows the field of view of the nerve cell under test and the light output port under test under an optical microscope.
  • Figure 4 shows the selection of auditory nerve cells to be tested in the imaging field after fluorescence annotation.
  • Figure 5 is a schematic diagram of the process of Fura-2 binding calcium ions and the excitation and emission spectra.
  • Fig. 6 shows the measurement results without external laser (light A) stimulating auditory nerve cells.
  • Fig. 6A shows the selected 6 cells to be tested, which are marked with numbers 1, 2, 3, 4, 5, 6 in the figure, and Fig. 6B To select the real-time ion concentration curve corresponding to the cell.
  • Figure 7 is the measurement result of the 450nm pulse laser (light A) stimulating the auditory nerve cells.
  • Figure 7A is the selected 6 cells to be tested, which are marked with numbers 1, 2, 3, 4, 5, 6 in the figure, and Figure 7B To select the real-time ion concentration curve corresponding to the cell.
  • Fig. 8 shows the measurement results of pulsed laser with wavelength 450nm ⁇ 808nm ⁇ 450nm sequentially stimulating auditory nerve cells.
  • Fig. 8A is the selected 6 cells to be tested, which are marked with numbers 1, 2, 3, 4, 5, 6 in the figure. 8B is a real-time ion concentration curve corresponding to selected cells.
  • the following embodiments are exemplary demonstrations of the measurement method of the present invention.
  • the method of the present invention is not specifically limited to the auditory nerve cells used in the following examples, and the cells may be any cells desired to be measured.
  • the light A in the present invention, as the pulse light to be measured is a fast-switching monochromatic light source with adjustable wavelength, light intensity, pulse width, repetition rate, and timing.
  • the general procedure of this example is as follows: the spiral ganglion cells in the cochlear axis of the C57-BL black mouse pups, which were provided by the Animal Experimental Center of Shandong University, were selected in a petri dish with cell slides.
  • the temperature of the petri dish is controlled at 37 ° C, the gas environment is 95% air and 5% CO 2 , DMEM-F12 medium specially used for nerve cell culture is added, and cultured in the dark environment for 24 hours. After that, the culture medium was removed, and the petri dish was washed twice with an HBSS solution capable of maintaining cell activity for a short period of time, and the buffer contained a large amount of Ca 2+ .
  • the specific wavelength laser corresponding to the specific fluorescent calcium indicator to excite the fluorescent label.
  • select cells with complete morphology and proper location distribution as shown in Figure 4.
  • the light intensity, pulse width, repetition rate, and timing of the laser output from the light source to be tested ie, light A
  • the laser application time be selected according to the output laser energy.
  • Multiple light sources with different wavelengths to be tested are freely switched by the laser conversion interface, and the laser is applied to the cells to be tested through the optical fiber for screening of photosensitization effects. Set the parameters of the light source A to be measured, and then select the excitation light B and C.
  • the excitation of the fluorescent probe starts to operate, and the output light wavelength (that is, the excitation light B and C) can be output for the absorption wavelength of different specific calcium ion fluorescence indicators.
  • the output light wavelength that is, the excitation light B and C
  • the output light wavelength can be output for the absorption wavelength of different specific calcium ion fluorescence indicators.
  • Meta Flour analysis software draws real-time fluorescence D intensity curve to reflect the change of calcium ion concentration of selected cells, operate as above, When the light source A is turned off, the curve reflects the real-time situation of the calcium ion concentration under the resting state of the cell or without external light stimulation, while the light source A is turned on, the curve reflects the real-time situation of the calcium ion concentration under the stimulation of external light.
  • the wavelength of the light source to be measured (that is, light A) should be kept at a difference of tens of nm from the absorption wavelength of the specific fluorescent calcium indicator (that is, excitation light B or C) and the fluorescence emission wavelength (that is, light D).
  • the difference should be kept above 40 nm in this embodiment. During the experiment, make sure that the position of the cell slide and the output fiber are always fixed.
  • the spiral ganglion cells in the cochlear axis of the C57-BL type black mouse pups born at seven days of birth provided by the Animal Experiment Center of Shandong University were selected and cultured in vitro in a culture dish with cell slides, the temperature of the culture dish was controlled at At 37 ° C, the gas environment is 95% air and 5% CO 2 , and DMEM-F12 medium specially used for nerve cell culture is added, and cultured in the dark environment for 24 hours. After that, the culture medium was removed, and the petri dish was washed twice with an HBSS solution capable of maintaining cell activity for a short period of time, and the buffer contained a large amount of Ca 2+ . Next, add the specific fluorescent calcium indicator Fura-2 and incubate for 30-60 minutes.
  • Fura-2 is currently the most commonly used calcium ion fluorescent indicator (also called calcium ion fluorescent probe), which belongs to the chemical calcium indicator in the indicator type and can be used with intracellular free calcium Ion-specific binding.
  • Fura-2 is excited by ultraviolet light, the excitation wavelength of the combined form Fura-2 is 340nm, the excitation wavelength of the free form Fura-2 is 380nm, as shown in Figure 5, and the emission spectrum peak is at 505-520nm, there is no significant change.
  • the ratio of the fluorescence intensity at the two excitation wavelengths that is, the ratio of F 340 / F 380
  • the ratio of calcium-bound Fura-2 to unbound Fura-2 can be determined, and the concentration of free calcium ions can be obtained using the Grynkiewicz formula .
  • the Grynkiewicz formula is expressed as follows:
  • K d is the equilibrium dissociation constant of the combination of Fura-2 and calcium ions, and its value is closely related to temperature, pH value, ion concentration, etc., it is 224 at 37 °C; ⁇ is zero intracellular calcium and saturated calcium at 380nm Fluorescence intensity ratio; R is the fluorescence intensity ratio of F 340 / F 380 at each measuring point; R min is the fluorescence intensity ratio of F 340 / F 380 at zero calcium; R max is the fluorescence intensity ratio of F 340 / F 380 at saturated calcium, R The max / R min value is between 13 and 25.
  • the intracellular Ca 2+ concentration of the selected cells is measured using the calcium ion imaging system.
  • fix the optical fiber of the light A to be measured with a three-dimensional adjuster that is, the aforementioned three-dimensional positioning bracket
  • the four parameters of light intensity, pulse width, repetition rate, and timing of the light source to be measured at different wavelengths need to be set.
  • the resting cells are excited with a fluorescent probe to collect the fluorescence intensity in the resting state, and the output light wavelength is switched between the two absorption wavelengths 340nm and 380nm of Fura-2.
  • Use the data analysis software to draw the real-time fluorescence intensity curve, reflecting the resting condition of the selected cell calcium ion concentration.
  • the light sources to be tested with three wavelengths of 450nm, 808nm and 1065nm can be freely switched by the laser conversion interface, and the laser light can be applied to the cells to be tested through the optical fiber for photosensitization. Screening measurement.
  • the experiment adopts grouped intermittent irradiation of laser signals, and each group of cells is only irradiated with a single wavelength of laser.
  • each group of cells is only irradiated with a single wavelength of laser.
  • the cells to be tested are cultured in a suitable dark environment to avoid quenching of cell fluorescence.
  • the changes in calcium ion concentration of nerve cells are observed as follows.
  • a 450nm pulsed laser is applied at a repetition rate of 11 Hz, a pulse width of 300 us, light intensity: gradually increased from zero, and calcium ion data acquisition speed: 2 ms / time (acquisition rate: 500 times / sec).
  • This phenomenon shows that the addition of a pulsed laser signal with a wavelength of 450 nm produces nerve impulses in animal auditory nerve cells and induces the transduction function of the auditory nerve. Observing the position of the cells and the optical fiber again, it can be found that the optical fibers of this group of experiments are placed on the upper right of the microscope field of view. Among the six cells selected in the microscope field of view, the cells closer to the fiber port (number 2, 3, 5, 6 ) The more obvious nerve impulses are generated, and the nerve cells far away from the optical fiber (number 1, 4), although the number of nerve impulses generated is basically the same, the amplitude of each impulse is smaller. This phenomenon may have a certain relationship with the output spot energy of the fiber port, because the spot is a circular surface with a diameter of 100 ⁇ m, and its illuminance or energy will gradually attenuate from the center of the circle to the surroundings.
  • the intracellular Ca 2+ concentration of the six nerve cells selected under the microscope field of vision did not change significantly before and after the laser injection. It can basically be considered that the auditory nerve cells did not produce the corresponding impulse.
  • the pulsed laser with a wavelength of 1065nm has no obvious effect on the transduction of nerve cells.
  • This phenomenon may be due to the fact that after a period of 808nm laser irradiation, although there is no significant change in calcium ions, the activity of nerve cells may be affected to some extent. When 450nm light is re-irradiated, the cell transduction response decreases.
  • the calcium ion imaging system uses the 340nm / 380nm acquisition light band and the fluorescence emission light band (505nm to 520nm) when collecting Ca 2+ concentration data. Although it is closer to the 450nm band of the laser to be measured, it is still more than 50nm. It can be considered that there is no obvious interference of the optical signal.

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Abstract

一种细胞特异性光敏效应的实时筛查与测量系统及方法，方法包括采用待测光A刺激荧光染色的待测细胞，采用光B和/或光C对细胞进行荧光激发，激发荧光D进一步通过CCD荧光成像，并传输至电脑进行数据处理及绘图，分析细胞离子浓度的实时变化判断光敏效应的存在或其响应情况；其中，待测细胞为离体细胞，细胞所处环境中存在待测离子且加入与待测离子相应的离子荧光指示剂；待测光A为多参数可调单色光源；光A、B、C、D波长之间相互间隔。方法快速、可重复，是一种普适的测量细胞光敏效应的方法。

Description

细胞特异性光敏效应的实时筛查与测量系统及方法 技术领域
本发明属于生物、医学及光电子学等多学科交叉领域，具体涉及细胞特异性光敏效应的实时筛查与测量系统及方法。
背景技术
自然界生物体细胞存在光敏感现象，研究已发现：神经细胞个体或群体的活动可用时间精确、快变、无创的光信号调控，这也是系统神经科学终极追求的目标。
生物细胞中含有各种门控的、离子选择性的由溶质浓度和跨膜电位差驱动的离子转运通道。离子通道的活性由通道蛋白的开或关两种构象通过应答各种刺激信号(如，光、电、热、机械、磁等)所调节。因此可通过信号刺激细胞上的离子通道来实现调节神经细胞信号转导的功能。离子通道根据其构成蛋白应答信号的不同，分为光敏感离子通道、电压力通道，配体门通道、压力激活通道。
迄今为止，已找到20余种光敏感蛋白，利用它们，光信号可调控细胞功能、组织分化以及动物行为等生命现象，并出现了新兴学科“光遗传学”(Optogenetics)。光遗传学技术是一种将光学与遗传学相结合的新兴的实验技术，其利用病毒载体将光敏感蛋白表达于可调控的靶细胞或器官组织上，采用特定参数的光信号调控细胞膜上光敏感通道的开启或关闭，进而对细胞生理功能精确调控，该技术已开始用于某些神经系统疾病的治疗研究。
光敏感通道是一种受光脉冲控制的具有跨膜结构的特异性或选择性离子通道，它可以快速形成光电流并使细胞发生电生理反应。例如，ChR2(Channelrhodopsin-2)光敏感通道蛋白是一种受光脉冲控制的具有跨膜结构的非选择性阳离子通道蛋白。该光敏感通道蛋白拥有可以快速形成光电流并使细胞发生去极化电生理反应。而钙离子是动物细胞内的第二信使，参与细胞的多种功能活动，如肌细胞的收缩、腺体的分泌、神经递质的释放、细胞分化和神经性死亡等等。这些重要的功能活动都伴有细胞内钙离子浓度的变化。但钙离子在细胞内通常是不可见的，钙离子成像技术就是根据钙离子浓度与神经细胞活动之间有严格的对应关系，利用钙离子可结合的荧光染料(即钙离子指示剂(calcium indicator))，将神经元细胞内钙离子浓度的变化通过荧光信号的变化记录下来，从而可监测神经细胞信号转导情况。例如，哺乳动物某神经元在静息状态下胞内钙离子浓度为 50-100nM，而当神经元活动时胞内钙离子浓度会显著上升。
除了ChR2外，HpHR(Halorhodopsin)是一种可抑制神经细胞兴奋的氯离子转运光敏蛋白，ArchT(Archaerhodopsin-T)是一种抑制性超极化质子泵。光敏感离子通道蛋白作为光调控技术的关键组成部分，对神经细胞的快速激发、快速抑制、双稳态调节等神经活动至关重要。
由于细胞光敏效应存在特异性，对光信号有选择性，故在各类有光调控效应的实验中，光敏效应特性是依赖激发光信号参量的，实验条件也各有差别，甚至在光控神经信号转导研究中(例如：神经回路的功能)，需要多路光信号同时刺激多个脑区的神经元。因此，在光控神经细胞信号转导研究中，需要有符合生物实验安全性要求的多通道、多参数可调的激发光，以构建细胞光敏效应筛查系统。
目前研究所用的光调控靶向细胞大多是通过病毒转染的转基因方式构建，而且调控用的激发光信号多在可见光波段。一方面转基因方式对未来该技术的疾病治疗存在不确定的安全性问题，另外更长波段激发光是否也可激发细胞光敏效应，以及哺乳类动物天然细胞上是否存在光敏效应，均是值得探索研究的。
目前，光遗传技术通过基因技术将光敏感通道蛋白基因移植入对应的生物细胞内，并使其表达产生光敏感通道蛋白是生物调控技术研究热点。经查询检索，有发明专利“可视化光刺激系统和可视化光刺激方法”(CN200910132986.7)利用蓝光(473nm)和黄光(593nm)对导入光敏蛋白的转基因细胞实现了光刺激和成像检验。但该专利对于刺激光的使用仅采用了两个波段的光，不能对待测细胞的更多光波段的敏感效应作检测。另外，该专利仅对于光遗传技术处理的转基因细胞进行成像实验检验，以证明其转基因的有效性，而无法对任意动物体细胞作光敏效应筛查，即其检测细胞类型是受限的。
自然界中有些细胞自身或经成分修饰后会含有光敏蛋白或结构，但这些光敏感结构对哪些波段的光有特异性响应、或某目标细胞是否有光敏效应、或若有光敏效应其对应的特异性光参量调控特性如何，对于这类分析检测，并没有一种广泛适用的方法来进行标定或测量。已有方法仅限于光敏效应结构或成分的表达验证，不具备快速查找或筛查的能力。随着细胞光敏效应及光遗传学技术研究的不断深入，亟需一种多光谱的、可操作性强的细胞特异性光敏效应的筛查方法，来加速发现自然细胞的特异性光敏效应以及监测光遗传技术中光敏效应的工作状况的研究。对此，本发明提出一种不限波段(可见及非可见)光信号、不再局限于转基因细胞、通用且可操作性强的细胞特异性光敏效应 实时筛查系统和方法，并能对筛选细胞的光敏特性进行定量与定位分析。
发明内容
本发明的目的在于提供一种实时筛查与测量细胞特异性光敏效应的方法，本发明的方法快速、可重复，可广泛应用于光遗传方向的生物技术以及生命科学和医学研究中，并且该方法可以从定量和定性两种层面分析光敏效应的工作状态。本发明可对待测细胞是否含有特定波长的光敏效应进行实时筛查，并进一步对光敏效应的工作情况实时测量；从而证实含有光敏效应的细胞对哪些波段的光有特异性反应。
其中，待测细胞既可以是光遗传技术处理后的转基因细胞亦可是普通的未经转基因技术处理的动植物细胞。待测细胞的刺激光波长可调，待测刺激光源可采用普照式的、或光纤耦合输出的、或LED发光器件等多种形式的单色光源。为精准测量其光敏效应，刺激光路输出端口既可加光束整形装置、也可自然端口输出。带有光束整形的光路便于能量更好的聚集、降低系统功耗，同时方便研究待测细胞的光敏反应的精准定位。而不带有光束整形的光路可以增加刺激光辐照面积，增加其光敏反应的范围，观察发散光不同位置光敏效应的反应效果。
本发明是通过如下技术方案实现的：
在本发明的第一方面，本发明提供一种实时筛查与测量细胞光敏效应的方法，所述方法包括采用待测光A刺激待测细胞，采用光B和/或光C激发细胞产生光D，对光D进行荧光成像，基于离子浓度的实时变化判断光敏效应的存在或进一步分析其工作情况；
其中，光A是一种波长(波段)、光强、脉宽、重复率、辐照时长(定时)等各参数在一定范围可任意调节的单色脉冲激光，以实现光信号的筛查。
待测细胞为离体活细胞，细胞所处环境中存在各种离子(待检测离子)，如钙离子(Ca 2+)，且加入离子荧光指示剂(或称为荧光探针)。比如，以钙离子浓度实时变化进行判断时，钙离子为待检测离子，加入的则为钙离子荧光指示剂或荧光探针。
所述方法通过离子成像技术检测荧光强度，并对数据进行处理，进而绘制实时或定时离子浓度变化的特异性曲线，判断光敏效应的存在或进一步分析其工作情况。
有别于其他方法，比如事先在细胞中导入特定光敏感基因表达特定光敏感蛋白，然后针对特定细胞的检测方法，本发明的方法并不特定细胞的种类和类型，因此，本发明的方法具有普适性。
所述方法中，所述光B和/或光C为离子荧光指示剂的激发光源；染色细胞(即加入 了离子荧光指示剂的待测细胞)受到激发会发射荧光，所发射的荧光为光D；
其中，所述光B和光C为离子荧光指示剂的激发光源时，离子荧光指示剂与游离离子结合后在光B下有特征激发，离子荧光指示剂不与游离离子结合时在光C下有特征激发；
如上所述，所述方法包括采用光A刺激待测细胞，采用光B和/或光C对细胞进行荧光成像，其中，采用光B和/或光C对细胞进行荧光成像时，是检测荧光D的强度，对荧光D进行成像。
如上所述，所述方法包括采用待测光A刺激荧光染色的待测细胞，采用光B和/或光C对细胞进行荧光激发，激发产生的荧光D进一步通过CCD荧光成像，并传输至电脑进行数据处理及绘图，分析游离离子浓度的实时变化判断光敏效应的存在或其响应情况。在CCD荧光成像的光路中，添加针对光B与光C的滤光片组，以消除激发光B与光C对于最终成像结果的影响；或在CCD荧光成像的光路中，添加只能使光D通过的滤光片，从而只对光D进行成像。
本发明所述的离子荧光指示剂是一类对特定离子(比如钙、钾、钠等离子)具有荧光特性的化学物质，其种类繁多，化学原理也不尽相同。就物理特性来看，主要是离子荧光指示剂的吸收波长和发射波长有所不同，即光B和或光C由吸收波长确定，光D是发射的荧光波长；部分荧光指示剂由单波长单色光激发，有的荧光指示剂还是由双波长单色光激发。表1总结了在使用几种常见钙离子荧光指示剂时光B和光C的建议波长。
表1
指示剂名称 K d值 光B建议波长 光C建议波长
Indo-1 230nM 405nm ------
Fura-2 140nM 340nm 380nm
Fluo-3 400nM 490nm ------
Fluo-4 345nM 490nm ------
BTC 7mM 400nm 480nm
Benzothiaza-1 660nM 340nm 380nm
当然，在使用其他特定钙离子荧光指示剂或其他离子荧光指示剂时可按照产品使用说明或指南确定光B和/或光C。
所述方法中，光A、光B、光C、光D的波长互不相同；其波段中心值必须有差距， 该差距至少在几十nm以上；所述几十nm比如为至少在10nm、15nm、20nm、25nm、30nm、40nm或50nm等等以上。
所述方法中，光A为单波段脉冲激光或者为包含有n个波段的N种可调参数的脉冲激光测量顺序的光的组合，其中，n、N为正整数，n≥2，N为n个波段的激光被测顺序的组合情况，一般N≥n。
在本发明的某些实施方式中，所述光A为包含有n个波段的N种脉冲激光测量顺序的光的组合时，采用光A刺激待测细胞为采用n个波段的脉冲激光按N种测量顺序分别刺激细胞。
所述方法中，所述n个波段的N种脉冲激光之间可自由切换；其中，在本发明的某些实施方式中，采用n个波段的N种脉冲激光自由切换分别刺激细胞时，相邻两次刺激的脉冲激光的波段不相同。
多个波段测量中，脉冲光的多参数可任意调节，所述参数如光强、脉宽、重复率、定时，参数的调节通常从小到大(或从弱到强)，每组n个波段测量中，各参数应保持一致，以保证测量的可比性。
所述方法中，所述相邻两次刺激的脉冲激光的波段中心值具有20～1000nm的差距，该差距优选为20～800nm，更优选为20～400nm，最优选为40nm。
比如，当光A为含有2个波长的光的组合或者在两种波长的光之间切换时，即n＝2，则N≥2；将两种波长的光定义为A 1和A 2，当N＝2时，则采用光A刺激细胞时，为采用A1和A2分别刺激细胞或交替刺激细胞，可以是采用A 1刺激细胞后，再采用A 2刺激细胞，或者采用A 1刺激细胞后切换为A 2刺激细胞，表示为A 1→A 2；也可以是采用A 2刺激细胞后再采用A 1刺激细胞，或者采用A 2刺激细胞后切换为A 1刺激细胞，表示为A 2→A 1；当N＝3或大于3时，采用光A刺激细胞可以是A 1→A 2→A 1或者A 2→A 1→A 2等等。
在某些较为具体的实施方案中，比如以钙离子浓度的实时变化实施本发明时，所述方法包括以下步骤：
(1)细胞处理：选取待测的细胞进行离体培养，单独加入钙离子和/或使用能够短时维持细胞活性的含有钙离子的溶液(比如HBSS溶液)冲洗培养皿；加入钙离子荧光指示剂或荧光探针后孵育；在光B和光C下选取有荧光标记的适宜细胞；
所述细胞离体培养方法可采用本领域技术人员常规掌握的方法；或者按照以下离体 细胞培养方法：选取细胞，在放有细胞爬片的培养皿中进行离体培养，培养皿温度控制在37℃，气体环境为95％空气和5％CO 2，加入适宜细胞培养的培养皿(比如，如培养神经细胞，则可选用专门用于神经细胞培养的DMEM-F12培养基)，培养24小时。
加入荧光指示剂或荧光探针后孵育可根据常规方法选择孵育时间，或者孵育30-60分钟。
所述钙离子荧光指示剂或荧光探针可以为比如Fura-2，或者其他如表1所示的钙离子荧光指示剂。
(2)钙离子荧光成像：分别通过光B和光C激发钙离子荧光指示剂，分别检测荧光强度；计算光B和光C两个激发波长上荧光强度的比率F B/F C，计算游离钙离子的浓度；
所述游离钙离子的浓度可根据Grynkiewicz公式计算得到；Grynkiewicz公式表达如下：
[Ca 2+] i＝K d×β×(R-R min)(R max-R)
其中，K d为荧光指示剂(比如Fura-2)和钙离子结合的平衡解离常数，其值与温度、pH值、离子浓度等密切相关，比如，Fura-2在37℃时为224；β是胞内零钙和饱和钙时在光C的荧光强度比；R是各测定点F B/F C荧光强度比值；R min是零钙时F B/F C荧光强度比值；R max是饱和钙时F B/F C荧光强度比值，R max/R min值在13到25之间。
(3)光A刺激下的钙离子荧光成像：在步骤(2)的方法的基础上，首先，固定光A的发射端，以确保光A和样品间的距离与入射角度。然后，使用光A刺激细胞，通过在成像光路上设置滤光片组仅通过光D，在进行光A刺激时，对光D进行钙离子荧光成像，记录其变化。当光A为非可见光波段时，需要首先使用可见光通过相同光路进行标记后，固定光A打入位置，更换光A为待测非可见光后，再进行荧光成像。最后，计算光A刺激下游离钙离子的浓度。
(4)根据步骤(2)和步骤(3)实时或定时得到的游离钙离子浓度绘制游离钙离子浓度变化曲线。比较离子浓度变化曲线上未经光A刺激与在光A刺激下的游离钙离子浓度的变化，判断光敏效应的存在或进一步分析其工作情况。
绘制游离钙离子浓度变化曲线可在步骤(2)或步骤(3)中直接进行以实现实时同步。最后再比较离子浓度变化曲线上未经光A刺激与在光A刺激下的游离钙离子浓度的变化，判断光敏效应的存在或进一步分析其工作情况。
在本发明的某一实施方式中，本发明对听神经细胞进行了检测，包括：采用光A刺激离体培养的听神经细胞(比如选取耳蜗蜗轴中的螺旋神经节细胞)，细胞所处环境中存在钙离子并加入钙离子荧光指示剂，采用光B和光C对细胞进行荧光成像，基于游离钙离子浓度的实时变化判断光敏效应的存在或进一步分析其工作情况。具体地，所述方法包括：
(1)细胞处理：选取听神经细胞(比如螺旋神经节细胞)进行离体培养，使用HBSS溶液冲洗培养皿(至少冲洗一次)；加入钙离子荧光指示剂，比如Fura-2后孵育30-60min；在340nm的紫外光和380nm的紫外光下选取有荧光标记的适宜细胞；所述适宜指在荧光标记的细胞成像图像中，选取形态完整、位置分布适当的细胞；
(2)钙离子荧光成像：分别通过340nm的紫外光或380nm的紫外光激发钙离子荧光指示剂，分别检测荧光强度，计算两个激发波长下的荧光强度的比率F 340/F 380，计算游离钙离子的浓度；
其中，游离钙离子浓度可根据Grynkiewicz公式计算得到。
Grynkiewicz公式表达如下：
[Ca 2+] i＝K d×β×(R-R min)(R max-R)
其中，K d为Fura-2和钙离子结合的平衡解离常数，其值与温度、pH值、离子浓度等密切相关，37℃时为224；β是胞内零钙和饱和钙时在380nm的荧光强度比；R是各测定点F 340/F 380荧光强度比值；R min是零钙时F 340/F 380荧光强度比值；R max是饱和钙时F 340/F 380荧光强度比值，R max/R min值在13到25之间。
(3)光A刺激下的钙离子荧光成像：在步骤(2)的方法的基础上，使用光A刺激细胞，并进行钙离子荧光成像，计算光A刺激下游离钙离子的浓度；所述光A为脉冲激光，其波长范围比如可以为450nm～1065nm中的任意波段或任意多个波段的组合；
采用光A刺激细胞，可以使用波长范围为450nm～1065nm中的任意波长(包括端值450nm和1065nm)的光分别或组合后在不同时刻分别照射细胞以给与刺激；
以组合光交替进行刺激时，相邻两次刺激的光的波长具有20～1000nm的波长差，该波长差优选为20～800nm，更优选为20～400nm，或40nm；更为精准的说法为，该波长差为波段中心值差。
比如，单独以450nm或808nm或1065nm的光刺激细胞；或者以选自450nm、808nm和1065nm的光中的至少两种波长的光的组合交替照射细胞，比如先后依次以 450nm→808nm→450nm脉冲激光刺激听神经细胞(即n＝2，N＝3)；
在一种实施方中，当光A为非可见光波段时，先试用可见光通过相同光路或位置进行标记后，固定光A打入位置，更换光A为待测非可见光后，再进行荧光成像。
(4)根据步骤(2)和步骤(3)实时或定时得到的游离钙离子浓度绘制游离钙离子浓度变化曲线；比较离子浓度变化曲线上未经光A刺激与在光A刺激下的游离钙离子浓度的变化，判断听神经细胞上光敏效应的存在或进一步分析其工作情况。
利用本发明的方法探究听神经光敏效应的存在，对于研究听神经在光刺激下的工作机制提供了重要依据，对于今后进一步研究光遗传技术在听神经的调控以及应用于临床神经系统疾病的研究具有重要意义。
在本发明的第二方面，本发明提供了一种实时筛查和测量细胞特异性光敏效应的系统，包括：
光源A，其发出光A刺激待测细胞；
离子成像系统，其至少包括荧光激发光路系统和CCD成像系统，荧光采集光路激发不同荧光，CCD成像系统检测荧光强度并进行图像数据采集；
以及，数据处理系统，其处理离子成像系统采集的数据。
进一步地，在本发明的实施方式中，光源A为多波段可切换、参数可调节的单色脉冲激光刺激光源，所述参数包括光强、脉宽、重复率和定时。
在本发明的实施方式中，光源A可发射可见光和非可见光。
在本发明的实施方式中，光源A与输出光路1耦合，可通过切换光源A和/或调节光源A的参数在不同时段输出光A。
在本发明的实施方式中，光源A可输出参数可调节的单波段脉冲激光或者可以N种切换顺序输出具有包含有n个波段的脉冲激光，其中，n、N为正整数，n≥2，N≥n。
在本发明的实施方式中，光源A为普照式的发光器件、光纤耦合输出的发光器件、或LED发光器件。
在本发明的实施方式中，光源A的发射端为带有光束整形的发射端口或不带有光束整形的自然发射端口。
在本发明的实施方式中，光源与光路的位置可调，位置包括光源或光路距离光源的远距离端口与带测点或待测区域间的距离和光A射向待测点或待测区域的角度。比如，其位置的调节可采用某种固定装置实现。
在本发明的实施方式中，离子成像系统中，荧光激发光路系统中至少包括B、C两种光源或者至少包括能够实现B、C两种光源的切换或共存的光源；
其中，光源B是荧光离子探针与对应离子结合时的特征激发波段光源，该特征激发光为光B；光源C是荧光离子探针与对应离子非结合时的特征激发波段光源，该特征激发光为光C；光源B和光源C与输出光路2耦合，交替输出光B、光C照射待测细胞，激发荧光D。
在本发明的实施方式中，离子成像系统中，CCD成像系统中设有观察光路，其观察光路上设有光片，该光片可为只能透过光D的光片或为能够同时阻断光A、光B和光C，防止A、B、C三种光透过的光片；CCD成像系统通过带有光片的观察光路检测荧光并进行图像数据采集，采集到的数据传输至数据处理系统。
在本发明的实施方式中，CCD成像系统采集的数据通过数据链路传输至数据处理系统，数据处理系统对数据进行分析、计算并绘图，可得到实时数据和图像。
更进一步的，在本发明的实施方式中，本发明所述实时筛查和测量细胞特异性光敏效应的系统包括：
(1)光源A(即待筛查光源)，光源A为多波段的可切换的脉冲激光刺激光源(所述不同波段间的调节或自由切换可通过比如激光转换接口或其他具有该功能的部件实现)，光源A可对包括光强、脉宽、重复率、定时四个参数进行调节；
光源A通过激光转换接口(比如法兰转换器)与输出光路1(比如光纤，在本发明中此处的光纤也被称为输出光纤1，直径为100μm)耦合及光源切换，可在不同时段产生不同波段的脉冲激光；光源A输出的激光通过输出光路1(比如输出光纤1，光纤与样品不接触)施加到待测样品上；光源A发光器件可以是光纤耦合的半导体激光器或LED(比如μLED)等等。光源A的发射端(也可称输出端)分为两种，即带光束整形的发射端口与不带光束整形的自然发射端口。带有光束整形的光路便于能量更好的聚集、降低系统功耗，同时方便研究待测细胞的光敏反应的精准定位，以及降低因调整光路位置而引起的影响，提高检测稳定性和灵敏性。而不带有光束整形的光路可以增加刺激光辐照面积，增加其光敏反应的范围，观察发散光不同位置的细胞光敏效应的反应效果。
待测样品为离体培养的细胞，细胞所处环境中存在待检测离子并已加入与待检测离子相应的离子荧光探针；
待测样品及光A输出端通过固定装置固定，比如离子成像系统的操作台上可以有盛 放待测样品的小皿及三维定位支架，该三维定位支架可固定待测样品(比如细胞爬片)和输出光纤；该固定装置也可以实现光A发射端与待测样品之间距离的固定，及光A的入射角度的选取；输出光纤和待测样品要保持合适的距离，在测量时务必确保细胞爬片和输出光纤的位置始终固定。
(2)离子成像部分，其包括荧光探针激发光路系统和CCD成像系统；
其中1)荧光探针激发光路系统，该光路至少包括B、C两种光源或者能够实现B、C两种光源的切换与共存，光源B和/或光源C为离子荧光指示剂的特征激发波段光源；通过光源B或光源C照射待测样品或者通过光源B和光源C交替照射待测样品(光通过输出光路2照射至待测样品，该输出光路2比如为输出光纤2)，激发不同荧光产生荧光强度，该受到激发产生的荧光为荧光D；
2)CCD成像系统，其通过观察光路(比如CCD)检测荧光强度并进行图像数据采集，将数据通过数据链路传输至数据处理系统；所述观察光路应包含宽频带可调波段滤光片组合，该滤光片可以为光A、光B与光C的过滤片，或者为只可透过荧光D的滤光片，从而消除光A、B和C的干扰，使被采集的荧光D通过CCD成像；
此外，所述观察光路还含有一条通向光学显微镜的观察光路支路。通过光学显微镜的观察目镜观察视野内的细胞和输出光纤位置。以便保证待测激光可以通过输出光路1(输出光纤1)准确辐照到待测细胞。该观察光路支路及与之相连的光学显微镜在确定好细胞和输出光纤位置后可移除或关闭。其本身并不直接参与光敏效应的实时筛查与测量。
或者采用集成有以上功能的其他装置，可以实现荧光强度的检测及图像数据的同步采集；
(3)数据处理系统，其对离子成像系统采集并同步传输的数据进行计算并绘图，得到相应的实时离子浓度曲线；
所述数据处理系统(比如数据处理电脑)可采用比如MetaFluor荧光比例成像软件，该软件可同步显示原始数据、比值图象、荧光强度曲线图、比率曲线图、离子浓度曲线图以及诸如明场或相差成像等非比例测定图像。软件可以同时成像和测量两种不同的比例测定指示剂而不受染料负载浓度、条件或发射强度的影响。
此外，需要说明的是，光的输出和输入都需要光路(比如光纤、光束整形、CCD)，光路与相应装置或部件的连接可选用本领域熟知的电路接口或转换接口实现。
在本发明的一种实施方式中，本发明所述系统如图2所示。
以及，本发明的第三方面，本发明提供了一种使用如上所示系统实时筛查和测量细胞光敏效应的方法，所述方法包括如下步骤：
(1)细胞处理：选取待测的细胞进行离体培养，单独加入待检测离子或使用能够短时维持细胞活性的含有待检测离子的溶液冲洗培养皿；加入与待检测离子相应的离子荧光指示剂后孵育；在光源B和/或光源C激发下选取有荧光标记的适宜细胞；
其中，细胞处理完成后，从培养皿中将细胞爬片取出，放入离子成像系统操作台的小皿中，通过调节三维定位支架，固定好细胞爬片和光A输出口位置，光A输出口和细胞要保持合适的距离，不能与细胞发生接触；为了避免荧光淬灭，整个过程要在暗光条件下进行。通过光学显微镜观察视野内的细胞和输出A光口位置。关闭光学显微镜的光，用特异性荧光离子指示剂所对应的特异性波长激光(通过荧光探针激发)进行荧光标记，在荧光标记的细胞成像图像中，选取形态完整、位置分布适当的细胞。
(2)离子荧光成像：分别通过荧光探针激发光路系统的光源B和/或光源C激发离子荧光指示剂，通过CCD成像系统分别检测荧光探针与待测细胞内游离离子以及结合离子发出的荧光强度静息值(即荧光D，如上所述)，并实时将数据传输至数据处理系统，计算实时或定时的无光源A刺激下的游离待测离子浓度；
(3)光源A刺激下的离子荧光成像：打开光源A刺激细胞，重复步骤(2)的操作，进行光源A刺激下的离子荧光成像，计算光源A刺激下的实时或定时的游离离子的浓度；
(4)CCD成像系统检测步骤(2)和步骤(3)中的荧光强度并将数据传输至数据处理系统；数据处理系统计算数据并绘制实时的离子浓度曲线；
(5)比较实时离子浓度变化曲线上未经光A刺激与在光A刺激下的游离离子浓度的变化，判断光敏效应的存在或进一步分析其工作情况。
在上述方法中，光源A同一时刻只生成特定参数的单波段脉冲激光，光源A可在不同时刻切换波段并随时调整参数，所述参数包括光强、脉宽、重复率和定时；
在检测过程中，光源A可生成包含有n个波段的N种脉冲激光测量顺序的组合，其中，n、N为正整数，n≥2，N≥n；所述n个波段的N种脉冲激光测量顺序之间可自由切换；
优选地，采用n个波段的N种脉冲激光自由切换在不同时刻分别刺激细胞时，相邻两次刺激的脉冲激光的波段不相同；
优选地，所述相邻两次刺激的脉冲激光的波段具有20～1000nm的差距，该差距优选为20～800nm，更优选为20～400nm，最优选为40nm。
优选地，光A、光B、光D和光C的波长互不相同；其中，在本发明的某些实施方式中，所述光A、光B、光D、光C的波长(波段中心值)相互之间差值至少在几十nm以上，比如10nm、15nm、20nm、25nm、30nm、35nm、40nm或50nm等等以上(可根据光源设备单色性而定)。
优选地，所述待检测离子比如钙离子，所述离子荧光指示剂比如选自表1中的任一种。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为本发明方法的原理示意图；其中，(a)为未经过特定波长的光照射的细胞内外离子状况；(b)为经过特定波长的光照射的细胞内外离子状况。
图2为本发明实时筛查和测量细胞特异性光敏效应的系统示意图。系统中包含3种光源，其中光源A(即待测光源)为待筛选波段可调光强、脉宽、重复率、定时等参数的脉冲激光。该光源由可调参数激光器或发光器件发射出相应参数的脉冲激光。生成的特定参数的脉冲光信号通过光转换接口与输出光路1耦合及光源切换，可在不同时段产生不同波段的脉冲光。该光信号通过输出光路1照射到对应的待测样品细胞上。光源B和光源C是荧光探针激发光路系统中的两个光源，分别是离子与探针结合、非结合时的特征激发光源。通过光源B和光源C交替通过输出光路2照射该待测样品细胞，激发染色后的待测样品，产生相应的荧光D，通过CCD成像系统进行图像数据采集，并通过数据链路传输至数据处理电脑，采用图像处理软件进行数据的计算和绘图，得到相应的实时离子浓度曲线。
图3为光学显微镜下的待测神经细胞和待测光输出口视野。
图4为荧光标注后成像视野中待测听神经细胞的选取情况。
图5为Fura-2结合钙离子的过程及激发光和发射光光谱示意图。
图6为无外来激光(光A)刺激听神经细胞的测量结果，其中，图6A为选取的6个待测细胞，在图中以编号1、2、3、4、5、6标示，图6B为选取细胞对应的实时离子浓度曲线。
图7为450nm脉冲激光(光A)刺激听神经细胞的测量结果，其中，图7A为选取的6 个待测细胞，在图中以编号1、2、3、4、5、6标示，图7B为选取细胞对应的实时离子浓度曲线。
图8为波长450nm→808nm→450nm脉冲激光顺序刺激听神经细胞的测量结果，其中，图8A为选取的6个待测细胞，在图中以编号1、2、3、4、5、6标示，图8B为选取细胞对应的实时离子浓度曲线。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
以下实施例为本发明测量方法的示例性展示，在以下实施例中，我们通过本发明的方法对听觉神经细胞进行测量发现听觉神经细胞中存在光敏效应，且该光敏效应对照射光的波长具有选择性。应当理解的是，本发明的方法并不特定的限定于下述实施例中采用的听觉神经细胞，该细胞可以是任意想要测定的细胞。且本发明中光A，作为待测脉冲光是一种波长、光强、脉宽、重复率、定时均可调节的快速切换单色光源。
该实施例的大致流程如下：选取出由山东大学动物实验中心提供的出生七天的C57-BL型黑鼠幼鼠耳蜗蜗轴中的螺旋神经节细胞，在放有细胞爬片的培养皿中进行离体培养，培养皿温度控制在37℃，气体环境为95％空气和5％CO 2，加入专门用于神经细胞培养的DMEM-F12培养基，黑暗环境下培养24小时。其后，撤掉培养基，使用能够短时维持细胞活性的HBSS溶液冲洗培养皿两遍，该缓冲液中含有大量Ca 2+。接下来，加入特异性荧光钙指示剂孵育30-60分钟。进行完前期处理后，从培养皿中将细胞爬片取出，放入钙离子成像系统操作台的小皿中，通过调节三维定位支架，在光学显微镜视野的辅助观察下，固定好细胞爬片和输出光纤的位置，光纤和细胞要保持合适的距离，不能与细胞发生接触，这里用到的光纤纤径为100μm。为了避免荧光淬灭，整个过程要在暗光条件下进行。通过光学显微镜观察视野内的细胞和光纤位置，如图3所示。关闭光学显微镜，用特异性荧光钙指示剂所对应的特异性波长激光激发进行荧光标记，在荧光标记的细胞成像图像中，选取形态完整、位置分布适当的细胞，如图4所示。待测光源(即光A)输出激光的光强、脉 宽、重复率、定时四个参数均可任意调节，为了避免待测细胞失活，我们建议根据输出激光能量合理选择激光施加时间。多个不同波长的待测光源由激光转换接口实现自由切换，通过光纤将激光施加到待测细胞进行光敏效应的筛查。将待测光源A参数设置完成，然后选择好激发光B、C，此时荧光探针激发开始运转，可以针对不同特异性钙离子荧光指示剂的吸收波长进行输出光波长(即激发光B和/或C)的切换或者多波长光同时输出，与此同时光源A关闭或开启，与此同时Meta Flour分析软件绘制出实时荧光D强度曲线反映选取细胞钙离子浓度的变化情况，按如上操作，光源A关闭时，曲线反映的是细胞静息状态或无外来光刺激下的钙离子浓度的实时情况，而光源A打开，曲线反应的是细胞接受外来光刺激下钙离子浓度的实时情况。为了避免多余的干扰，待测光源波长(即光A)应与特异性荧光钙指示剂的吸收波长(即激发光B或C)和荧光发射波长(即光D)保持几十nm的差值。例如，为了保证实验的准确性，本实施例中该差值应保持在40nm以上。在实验时务必确保细胞爬片和输出光纤的位置始终固定。
实施例
1、细胞处理
选取由山东大学动物实验中心提供的出生七天的C57-BL型黑鼠幼鼠耳蜗蜗轴中的螺旋神经节细胞，在放有细胞爬片的培养皿中进行离体培养，培养皿温度控制在37℃，气体环境为95％空气和5％CO 2，加入专门用于神经细胞培养的DMEM-F12培养基，黑暗环境下培养24小时。其后，撤掉培养基，使用能够短时维持细胞活性的HBSS溶液冲洗培养皿两遍，该缓冲液中含有大量Ca 2+。接下来，加入特异性荧光钙指示剂Fura-2孵育30-60分钟。进行完前期处理后，从培养皿中将细胞爬片取出，放入钙离子成像操作台的小皿中，通过调节三维定位支架，固定好细胞爬片和输出光纤的位置，光纤和细胞要保持合适的距离和角度，不能与细胞发生接触，这里用到的光纤纤径为100μm。为了避免荧光淬灭，整个过程要在暗光条件下进行。通过光学显微镜观察视野内的细胞和光纤位置，如图3所示。关闭光学显微镜，用Fura-2所对应的吸收波长340nm和380nm的激光进行荧光标记，在荧光标记的细胞成像图像中，选取形态完整、位置分布适当的细胞，如图4所示。然后进行后续的测试实验。
2、钙离子荧光成像
钙离子荧光指示剂工作原理：Fura-2是目前最常用的一种钙离子荧光指示剂(也称钙离子荧光探针)，在指示剂类型中属于化学钙指示剂，可与胞内游离钙离子特异结合。Fura-2 由紫外光激发，结合形式的Fura-2激发波长为340nm，游离形式的Fura-2激发波长为380nm，如图5所示，而发射光谱峰在505-520nm，没有明显变化。因此通过检测两个激发波长上荧光强度的比率，即F 340/F 380比值，就可以确定结合钙的Fura-2与未结合Fura-2的比例，进而利用Grynkiewicz公式求出游离钙离子的浓度。Grynkiewicz公式表达如下：
[Ca 2+] i＝K d×β×(R-R min)(R max-R)
其中，K d为Fura-2和钙离子结合的平衡解离常数，其值与温度、pH值、离子浓度等密切相关，37℃时为224；β是胞内零钙和饱和钙时在380nm的荧光强度比；R是各测定点F 340/F 380荧光强度比值；R min是零钙时F 340/F 380荧光强度比值；R max是饱和钙时F 340/F 380荧光强度比值，R max/R min值在13到25之间。
钙离子成像系统：细胞处理完成后，使用钙离子成像系统对选定细胞胞内Ca 2+浓度进行测量。在测量前，用三维调节仪(即前述的三维定位支架)将待测光A的光纤进行固定，以确保实验中光A发射端与样品之间的距离和角度。实验中，务必确保细胞爬片和输出光纤的位置始终固定。
施加待测光开始前，还需对不同波长待测光源的光强、脉宽、重复率、定时四个参数进行设置。在未施加待测光A时，先对静息细胞用荧光探针激发光路系统采集静息状态下的荧光强度，在Fura-2两个吸收波长340nm和380nm之间进行输出光波长的切换，用数据分析软件绘制出实时荧光强度曲线的情况，反映选取细胞钙离子浓度的静息情况。再对听神经细胞施加光A的辐照时，450nm、808nm、1065nm三个波长的待测光源，由激光转换接口实现不同波段激光的自由切换，通过光纤将激光施加到待测细胞，进行光敏效应的筛查测量。
3、光A刺激下的钙离子荧光成像
无外来激光刺激的测量结果(即不施加光A刺激)
当无外来激光信号照射时，对细胞样品进行钙离子浓度采集(2ms/次)。如图6所示，在显微成像视野下选择的六个神经细胞，其胞内Ca 2+浓度没有很明显的变化，可以认为，此时听神经细胞处于静息状态，该测试结果可作为后续有外来激光刺激时细胞反应的参照。
单一波长激光刺激听神经细胞实验
考虑到动物细胞对于能量激光的耐受力，避免长时间高强度激光刺激可能使细胞失活，实验采取分组间歇辐照激光信号，每一组细胞只照射单一波长的激光。为了保证细胞活性，每做完一组测试，再更换另一组新的细胞进行后续实验。待实验的细胞，培养在适宜的黑 暗环境下，以避免细胞荧光淬灭。对于三种单一波长的激光刺激，神经细胞的钙离子浓度变化观测情况如下。
(1)450nm脉冲激光刺激的测量结果
施加450nm脉冲激光，重复率11Hz，脉宽300us，光强：由零逐渐提升，钙离子数据采集速度：2ms/次(采集率：500次/秒)。
启动外加光信号(对应图7B图下方白线)，持续照射中，测得明显的细胞内Ca 2+浓度明显变化，如图7所示，仔细观察会发现，随时间推移，Ca 2+浓度反应峰值有呈逐渐上升的趋势，直到达到某最大临界点后，Ca 2+浓度峰值才逐渐减小。
该现象说明，外加波长450nm的脉冲激光信号使动物听神经细胞产生了神经冲动，诱发出听神经的转导功能。再观察细胞与光纤位置，可以发现，该组实验的光纤放置在显微镜视野图的右上方，在显微镜视野中选取的六个细胞中，越靠近光纤口的细胞(编号2、3、5、6)产生的神经冲动越明显，而远离光纤的神经细胞(编号1、4)，尽管产生神经冲动的次数基本一致，但每一次冲动的幅度要小些。该现象可能与光纤端口输出光斑能量有一定关系，因为光斑是一个直径为100μm圆面，其照度或能量会由圆心向四周逐渐衰减。
(2)808nm脉冲激光刺激结果
更换另一组细胞，设定相同参数，外加808nm激光照射，在激光射入的相同时段内，在显微镜视野下选择的六个神经细胞胞内Ca 2+浓度没有明显的变化(如图6A所示)，虽然有一两个细胞内Ca 2+浓度呈轻微上升的趋势，但是通过对纵坐标数值进行量化发现浓度增量较总浓度量比值低于3‰，基本可以认为听神经细胞未产生冲动。实验表明，波长808nm的脉冲激光未对神经细胞转导产生明显影响。
(3)1065nm脉冲激光刺激结果
同上，将1065nm激光照射后，在激光射入的前后时刻，在显微镜视野下选择的六个神经细胞胞内Ca 2+浓度也没有明显的变化，基本可认为听神经细胞未产生相应冲动。实验表明，波长1065nm的脉冲激光未对神经细胞转导产生明显影响。
切换不同波长激光刺激实验
在单一波长激光对听神经细胞刺激测试中，每次采用不同组测试。为了排除不同组细胞可能存在细胞活性的差异，采用同一组细胞先后切换照射不同波长激光信号，进一步探究动物听神经细胞对激光波长选择性的反应。
本实验在450nm和808nm两个波长激光之间做450nm→808nm→450nm切换，测量结 果如图8所示，可以发现，在照射450nm激光时(时间轴对应第一个小白柱，即横坐标轴线上从左侧起第一个小白柱)，选取的六个细胞，胞内Ca 2+浓度均产生很明显的变化；紧接着关闭激光信号，改变光源波长参数为808nm，其它参数不变，再照射(时间轴对应第二个小白柱，即横坐标轴线上从左侧起第二个小白柱)，可以发现，在照射较长时间里，未见胞内Ca 2+浓度出现明显变化；之后，关闭808nm激光器，将激光波长再调回450nm照射(时间轴对应第三个小白柱，即横坐标轴线上从左侧起第三个小白柱)，可以看到，选取的六个细胞中只有一个细胞胞内Ca 2+浓度明显上升，但产生冲动幅度较之前照射450nm激光时细胞钙离子浓度反应明显变弱。该现象可能与经过一段时间808nm激光的照射后，尽管未发生钙离子明显变化，但神经细胞活性可能受到一定影响，当450nm光再照射时，细胞转导反应下降了。
实验结果：选择450nm、808nm和1065nm三种不同波长的脉冲激光对动物听神经细胞刺激，结果证实，450nm的激光能够使细胞内Ca 2+浓度产生显著改变，而且发现，光纤输出端口位置与听细胞刺激反应程度有关联关系；而另外两种波长(808nm，1065nm)的激光刺激未能对听神经细胞内Ca 2+浓度产生明显改变。由此初步证实，在适宜波长激光信号辐照下听细胞能产生神经冲动，有阳离子转导现象，或者说，激光诱发听神经反应具有波长选择性。
进一步，切换不同波长激光(450nm、808nm)刺激听神经细胞，结果证实，听神经细胞对于450nm光反应确实灵敏，重复引发钙离子浓度明显变化，能引发神经冲动或转导反应，而非敏感波段激光刺激时，细胞无明显神经冲动反应。
实验初步证实，能诱发离体听神经细胞钙离子浓度变化、从而引发神经转导功能的光刺激具有波长选择性，450nm是其敏感波段，更易激发神经转导所需的递质释放。该转导现象，从光遗传学角度推断，听神经细胞上应该存在对应450nm的光敏效应。
利用钙离子成像技术测得450nm波段激光引起细胞内Ca 2+浓度的明显变化现象，可得出，听神经细胞膜上有相关的光敏效应存在，细胞外液中的Ca 2+出现了大量内流，导致细胞内的Ca 2+浓度急剧上升；在移除450nm激光后，细胞内的Ca 2+浓度逐渐下降，这一现象与细胞电生理测量的一般现象是一致的。
此外，钙离子成像系统在采集Ca 2+浓度数据时所使用的340nm/380nm采集光波段和荧光发射光波段(505nm至520nm)，距离外加待测激光450nm波段尽管较近，但仍有50nm以上的间距，故可认为并未产生光信号的明显干扰。

Claims (10)

  1. 一种实时筛查和测量细胞特异性光敏效应的方法，所述方法包括采用待测光A刺激待测细胞，采用光B和/或光C激发细胞进行荧光成像，基于细胞内游离离子浓度的实时变化判断光敏效应的存在或进一步分析其工作情况；
    其中，待测光A是一种参数可调节的单色脉冲激光，所述参数包括波长、光强、脉宽、重复率、定时，光A为可见光或非可见光；
    待测细胞为离体活细胞，细胞所处生存环境中存在待检测离子且加入与待检测离子对应的离子荧光指示剂。
  2. 根据权利要求1所述的方法，其特征在于，所述方法通过离子成像技术检测荧光强度，并对数据进行处理，进而绘制实时或定时离子浓度变化的特异性曲线，判断细胞特异性光敏效应的是否存在或进一步分析其工作情况。
  3. 根据权利要求1或2所述的方法，其特征在于，光A为单波段脉冲激光或者为包含有n个波段的N种脉冲激光测量顺序的组合，其中，n、N为正整数，n≥2，N≥n；
    优选地，所述光A为包含有n个波段的N种脉冲激光测量顺序的组合时，采用光A刺激待测细胞为采用n个波段的脉冲激光按N种测量顺序分别刺激待测细胞；
    优选地，所述n个波段的N种脉冲激光之间可自由切换；
    优选地，采用n个波段的N种脉冲激光自由切换分别刺激细胞时，相邻两次刺激的脉冲激光的波段不相同；
    优选地，所述相邻两次刺激的脉冲激光的波段中心值具有20～1000nm的差距，该差距优选为20～800nm，更优选为20～400nm。
  4. 根据权利要求1或2所述的方法，其特征在于，所述光B和/或光C为离子荧光指示剂的激发光源；待测细胞受到激发会发射荧光，所发射的荧光为光D；
    优选地，所述光B和光C为离子荧光指示剂的激发光源，与荧光指示剂分子结合的离子(即结合离子)在光B下有特征激发，未与荧光指示剂分子结合的离子(即游离离子)在光C下有特征激发；
    优选地，采用光B和/或光C对细胞内离子进行激发时，可产生特征荧光D的发射，检测荧光D的强度，对荧光D进行成像。
  5. 根据权利要求1或2所述的方法，其特征在于，光A、光B、光C和光D的波长互不相同；
    优选地，所述光A、光B、光C和光D相互间的波长差值在10nm以上。
  6. 根据权利要求1至5中任一项所述的方法，其特征在于，所述方法包括：
    (1)细胞处理：选取待测的细胞进行离体培养，单独加入待检测离子或使用能够短时维持细胞活性的含有待检测离子的溶液冲洗培养皿；加入与待检测离子相应的离子荧光指示剂后孵育；在光B和/或光C下选取有荧光标记的适宜细胞；
    (2)离子荧光成像：分别通过光B和/或光C激发离子荧光指示剂，分别检测荧光强度；计算光B和光C两个激发波长上荧光强度的比率F B/F C，计算细胞内离子的浓度；
    (3)光A刺激下的离子荧光成像：根据步骤(2)的方法，使用光A刺激细胞，并进行光A刺激下的离子荧光成像；计算光A刺激下细胞内离子的浓度变化；
    (4)根据步骤(2)和步骤(3)实时或定时得到的离子浓度绘制离子浓度变化曲线；比较离子浓度变化曲线上未经光A刺激与在光A刺激下的离子浓度的变化，判断待测光A光敏效应的存在或进一步分析其工作情况；
    优选地，当光A为非可见光波段时，先试用可见光通过相同光路或位置进行标记后，固定光A打入位置，更换光A为待测非可见光后，再进行光A刺激时的荧光成像。
  7. 一种实时筛查和测量细胞特异性光敏效应的系统，所述系统包括：光源A，其发出光A刺激待测细胞；离子成像系统，其至少包括荧光激发光路系统和采集发射荧光的CCD成像系统，荧光激发光路激发不同离子的荧光，CCD成像系统检测荧光强度并进行图像数据采集；数据处理系统，其处理离子成像系统采集的数据。
  8. 根据权利要求7所述的系统，其特征在于，光源A为多波段可切换、参数可调节的单色脉冲光刺激光源，所述参数包括光强、脉宽、重复率和定时；
    优选地，光源A可发射可见光和非可见光；
    优选地，光A与输出光波段耦合，可通过切换光源波段(1,2,3……n)和/或调节光源A的参数在不同时段输出光A；
    优选地，光源A可输出参数可调节的单波段脉冲激光或者可以N种切换顺序组合输出具有包含有n个波段的脉冲激光，其中，n、N为正整数，n≥2，N≥n；
    优选地，光源A为普照式的发光源、器件、光纤耦合输出的发光器件、或LED发光器件；
    优选地，光源A的发射端为带有光束整形的发射端口或不带有光束整形的自然发射端口；
    优选地，光源与光路的位置可调，位置包括光源或光路距离光源的远距离端口与带 测点或待测区域间的距离和光A射向待测点或待测区域的角度。
    优选地，离子成像中，荧光激发光路系统中至少包括B和/或C两种光源或者至少包括能够实现B、C两种光源的切换或共存的光源；
    其中，光源B是荧光探针与对应离子结合时的特征激发波段光源，该特征激发光为光B；光源C是荧光离子探针与对应离子非结合时的特征激发波段光源，该特征激发光为光C；光源B和光源C与输出光路2耦合，交替输出光B、光C照射待测细胞，激发荧光D；
    优选地，离子成像系统中，CCD成像系统中设有观察光路，其观察光路上设有滤光片，该光片可为只能透过光D的滤光片或为能够同时阻断光A、光B和光C，防止A、B、C三种光透过的滤光片；CCD成像系统通过带有滤光片的观察光路检测荧光并进行图像数据采集，采集到的数据传输至数据处理系统；
    优选地，CCD成像系统采集的数据通过数据链路传输至数据处理系统，数据处理系统对数据进行分析、计算并绘图，可得到实时数据和图像。
  9. 一种实时筛查和测量细胞特异性光敏效应的方法，所述方法基于权利要求7或8中所述的系统，包括进行如下步骤：
    (1)细胞处理：选取待测的细胞进行离体培养，单独加入待检测离子或使用能够短时维持细胞活性的含有待检测离子的溶液冲洗培养皿；加入待检测离子的荧光指示剂后孵育；在光源B和/或光源C下选取有荧光标记的适宜细胞；
    (2)离子荧光成像：分别通过光B和/或光C激发离子荧光指示剂，CCD成像系统同步检测荧光并采集图像信息，该信息实时传输至数据处理系统；
    (3)光A刺激下的离子荧光成像：打开光源A刺激细胞，重复步骤(2)的操作，进行在光A刺激下的细胞内离子荧光成像，CCD成像系统同步检测荧光并采集图像信息，该信息实时传输至数据处理系统；
    优选地，当光A为非可见光波段时，先试用可见光通过相同光路或位置进行标记后，固定光A打入位置，更换光A为待测非可见光后，再进行荧光成像；
    (4)数据处理系统对实时接收到的步骤(2)和步骤(3)的信息进行分析、计算，并绘制实时离子浓度曲线图；
    (5)比较实时离子浓度变化曲线上未经光A刺激与在光A刺激下的离子浓度的变化，判断光敏效应的存在或进一步分析其工作情况。
  10. 根据权利要求9所述的方法，其特征在于，光源A可在不同时刻切换波段并随时调整参数，所述参数包括光强、脉宽、重复率和定时，但是，光源A同一时刻只生成特定参数的单波段脉冲光；
    在检测过程中，光源A可生成包含有n个波段的N种脉冲激光测量顺序的组合，其中，n、N为正整数，n≥2，N≥n；所述n个波段的N种脉冲激光之间可自由切换；
    优选地，采用n个波段的N种脉冲激光自由切换在不同时刻分别刺激细胞时，相邻两次刺激的脉冲光的波段不相同；
    优选地，所述相邻两次刺激的脉冲光的波段中心值具有20～1000nm的差距，该差距优选为20～800nm，更优选为20～400nm；
    优选地，光A、光B、光C、光D的波段中心值互不相同；优选地，，所述光A、光B、光C、光D相互之间的波段中心值差值至少在10nm以上。
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