AU2019368519B2

AU2019368519B2 - System and method for real-time screening and measurement of cellular specific photosensitive effect

Info

Publication number: AU2019368519B2
Application number: AU2019368519A
Authority: AU
Inventors: Lan TIAN
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2018-10-26
Filing date: 2019-10-25
Publication date: 2022-11-03
Anticipated expiration: 2039-10-25
Also published as: AU2019368519A1; CN111103272B; WO2020083398A1; CN111103272A

Abstract

A real-time screening and measurement system for a cell-specific photosensitive effect and a method thereof. The method comprises: stimulating fluorescence-stained cells to be detected with light A to be detected, using light B and/or light C to perform a fluorescence excitation on the cells, further subjecting the excitation fluorescence D to the fluorescence imaging by means of CCD, and transferring the image to a computer for data processing and graphing, and analyzing the real-time change of the concentration of ions in the cells to judge the presence of the photosensitive effect or the response thereof, wherein the cells to be detected are ex vivo cells, and there are ions to be detected in the environment where the cells exist, and ion fluorescence indicators corresponding to the ions to be detected are added to the environment; the light A to be detected is a multiparameter-adjustable monochromatic light source; and the wavelengths of lights A, B, C and D are spaced apart from each other. The method is fast and repeatable, and is a universal method for measuring the photosensitive effect of cells.

Description

SYSTEM AND METHOD FOR REAL-TIME SCREENING AND MEASUREMENT OF CELLULAR SPECIFIC PHOTOSENSITIVE EFFECT

Field of the Invention

The present invention belongs to the interdisciplinary fields of biology, medicine and

optoelectronics, and specifically relates to a system and method for real-time screening and

measurement of a cellular specific photosensitive effect.

Background of the Invention

Biological cells in the nature have photosensitivity. Studies have found that the activities

of an individual or group of nerve cells can be controlled by time-accurate, fast-changing, and

non-invasive optical signals, which is also the ultimate goal of systems neuroscience.

The biological cells contain various gated and ion-selective ion transport channels driven

by solute concentration and transmembrane potential difference. The activities of ion channels

are adjusted by the on or off conformation of channel proteins in response to various stimulus

signals (such as light, electricity, heat, machinery, and magnetism). Therefore, the signal

transduction function of nerve cells can be adjusted through signal stimulation of ion channels

on the cells. The ion channels are divided into photosensitive ion channels, voltage-sensitive

ion channels, ligand-gate ion channels, and stress-activated ion channels according to their

different constituent protein response signals.

So far, many photosensitive proteins have been found. With the photosensitive proteins,

optical signals can adjust life phenomena such as cell function, tissue differentiation and

animal behaviors, and optogenetics has emerged. The optogenetics is an emerging

experimental technology that combines optics and genetics. It uses viral vectors to express

photosensitive proteins on adjustable target cells or organ tissues, and uses optical signals of

specific parameters to adjust the opening or closing of photosensitive channels on cell

membranes, thereby accurately adjusting the physiological functions of cells. This technology

has begun to be used in the treatment research of some neurological diseases.

The photosensitive ion channel is a specific or selective ion channel with a transmembrane structure controlled by light pulses. It can quickly form photocurrent and cause an electrophysiological reaction in cells. For example, ChR2 (Channelrhodopsin-2) photosensitive channel protein is a non-selective cation channel protein with a transmembrane structure controlled by light pulses. The photosensitive channel protein has the ability to rapidly form photocurrent and cause a depolarizing electrophysiological reaction in cells.

Calcium ions are second messengers in animal cells and participate in multiple functional

activities of cells, such as muscle cell contraction, gland secretion, neurotransmitter release,

cell differentiation and neurological death. These important functional activities are

accompanied by changes in the intracellular calcium ion concentration. However, calcium ions

are usually invisible in cells. Calcium imaging technology is to record changes in the calcium

ion concentration in neuron cells through the changes in fluorescence signals based on the

strict correspondence between calcium ion concentration and nerve cell activity by using a

fluorescent dye that calcium ions can bind to (i.e. a calcium indicator), thereby monitoring the

signal transduction of nerve cells. For example, the intracellular calcium ion concentration of a

neuron in a mammal is 50-100 nM in a resting state, and the intracellular calcium ion

concentration will increase significantly when the neuron is active.

In addition to ChR2, HpHR (Halorhodopsin) is a chloride ion transport photosensitive

protein that can inhibit nerve cell excitement, and ArchT (Archaerhodopsin-T) is an inhibitory

hyperpolarized proton pump. As a key component of photoregulation technology,

photosensitive ion channel proteins are essential for neural activities such as rapid excitation,

rapid inhibition, and bistable regulation of nerve cells.

Due to the specificity of cell photosensitivity and selectivity to optical signals, the

characteristics of photosensitivity are dependent on the parameters of excitation optical signals

in various experiments with photoregulation effects, the experimental conditions are also

different, and even in transduction research of light-controlled nerve signals (for example: the

function of neural circuits), multi-channel optical signals are required to stimulate neurons in

multiple brain regions at the same time. Therefore, in the research of transduction of

light-controlled nerve cell signals multi-channel and multi-parameter adjustable excitation

light that meets safety requirements of biological experiments is required to construct a cellular photosensitivity screening system.

At present, most of the photoregulation targeting cells used in the research are constructed

by virus transfected transgenosis, and the excitation optical signals used for regulation are

mostly in visible light bands. On the one hand, the transgenosis has uncertain safety issues for

future disease treatments of this technology. On the other hand, whether excitation light in

longer bands can also stimulate cell photosensitivity, and whether mammalian natural cells

have photosensitivity, are worth exploring and studying.

At present, it is a research hotspot of biological regulation technology that the

optogenetics transplants photosensitive channel protein genes into corresponding biological

cells through gene technology, and causes same to express and produce photosensitive channel

proteins. Upon query and search, the invention patent entitled Visualized Light Stimulation

System and Visualized Light Stimulation Method (CN200910132986.7) uses blue light (473

nm) and yellow light (593 nm) to achieve light stimulation and imaging tests on transgenic

cells introduced with photosensitive proteins. However, this patent uses only two bands of

stimulation light, and cannot detect the sensitive effects of more light bands of target cells. In

addition, this patent only conducts imaging tests on transgenic cells treated by optogenetics to

prove the effectiveness of their transgenes, and cannot screen any animal somatic cells for

photosensitive effects, that is, detected cell types are limited.

In the nature, some cells contain photosensitive proteins or structures by themselves or

after component modification, but there is no widely applicable method for calibration or

measurement on the analysis and detection about which bands of light these photosensitive

structures have specific response, or whether a target cell has a photosensitive effect, or how

the regulation characteristics of specific optical parameters corresponding to the target cell are

if the target cell has the photosensitive effect. Existing methods are limited to the expression

verification of a photosensitive effect structures or components, and do not have quick

searching or screening abilities. With the continuous in-depth research of cell photosensitive

effects and optogenetics, a multi-spectral and highly operable detection method for a cellular

specific photosensitive effect is urgently needed to accelerate studies on the discovery of

specific photosensitive effects of natural cells and the monitoring of the working status of a photosensitive effect in optogenetics. In this regard, the present invention proposes a real-time screening system and method for cellular specific photosensitive effects, which are applicable to any bands (visible and invisible) of optical signals, are no longer limited to transgenic cells, are universal and highly operable, and can implement quantitative and positioning analysis on the photosensitivity of target cells. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

It is an object of the present invention to overcome or ameliorate at least one of the

disadvantages of the prior art, or to provide a useful alternative. Unless the context clearly requires

otherwise, throughout the description and the claims, the words "comprise", "comprising", and the

like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that

is to say, in the sense of "including, but not limited to".

Summary of the Invention

Provided a method for real-time screening and measurement of a cellular specific

photosensitive effect. The method of the present invention is fast and reproducible, and can be

widely used in optogenetic biotechnology, life science and medical research, and the method

can analyze the working status of a photosensitive effect from both quantitative and qualitative

levels. The present invention can detect whether target cells contain a photosensitive effect of a

specific wavelength, and further measure the working status of the photosensitive effect in real

time, thereby confirming that the cells including the photosensitive effect have specific

response to which bands of light.

The target cells may be transgenic cells treated by optogenetics or ordinary animal and

plant cells not treated by transgenosis. The wavelength of stimulation light for the target cells

is adjustable, and the stimulation light to be measured may be monochromatic light in multiple

forms of general illumination, fiber coupled output, or LED light-emitting devices. In order to

accurately measure the photosensitive effect, a beam shaping device can be added to an output

port of a stimulation light path, and the stimulation light can also be outputted from a natural port output. The light path with beam shaping facilitates better concentration of energy, reduces system power consumption, and facilitates the study of precise positioning of the photosensitivity of the target cells. The light path without beam shaping can increase the irradiation area of the stimulation light, increase the range of its photosensitivity, and observe responses of photosensitive effects of divergent light at different positions.

In the first aspect of the present invention, the present invention provides a method for

real-time screening and measurement of a cellular photosensitive effect, the method including

using light A to be measured to stimulate target cells, using light B and/or light C to excite the

cells to produce light D, performing fluorescence imaging on light D, and determining the

existence of a photosensitive effect based on the real-time change of ion concentration or

further analyzing the working status thereof;

wherein light A is a monochromatic pulsed laser whose parameters such as wavelength

(band), intensity, pulse width, repetition rate, and irradiation duration (time) can be adjusted

arbitrarily within certain ranges to achieve screening of optical signals.

The target cells are living cells in vitro, there are various ions (ions to be detected) in the

environment where the cells are located, such as calcium ions (Ca2+), and a fluorescent ion

indicator (or called a fluorescent probe) is added. For example, when measuring the real-time

change of calcium ion concentration, calcium ions are the ions to be detected, and a fluorescent

calcium indicator or probe is added in the environment where the cells are located.

The method detects fluorescence intensity through ion imaging technology, processes

data, then draws a specific curve of real-time or timed ion concentration changes, and

determines the existence of a photosensitive effect or further analyzes the working status

thereof.

Different from other methods, such as introducing specific photosensitive genes into cells

in advance to express specific photosensitive proteins and then detecting specific cells, the

method of the present invention does not specify the type of cells. Therefore, the method of the

present invention is universal.

In the method, the light B and/or light C are excitation light for the fluorescent ion

indicator; stained cells (i.e., target cells with the fluorescent ion indicator added) emit fluorescence when excited, and the emitted fluorescence is light D;

When the light B and the light C are excitation light for the fluorescent ion indicator, the

fluorescent ion indicator is characteristically excited under the light B after binding to free

ions, and the fluorescent ion indicator is characteristically excited under the light C when not

binding to free ions;

As mentioned above, the method includes using light A to stimulate target cells, 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 intensity of fluorescence D

is detected, the fluorescence D is imaged.

The concentration of free calcium ions can be calculated according to formulas known in

the art. For example, when light B and light C are used for fluorescence excitation, the

intensities of fluorescence D generated by the excitation are respectively detected (the

fluorescence intensities corresponding to light B and light C are denoted by FB and FC), and

the concentration of free calcium ions can be calculated based on a Grynkiewicz formula by

calculating the ratio FB/FC of the fluorescence intensities on the two excitation wavelengths of

light B and light C;

The Grynkiewicz formula is expressed as follows:

[Ca2+]j=Kdxpx(R - Rmin)/(Rmax - R)

Where, Kd is an equilibrium dissociation constant of binding between a fluorescent

indicator (such as Fura-2) and calcium ions, and its value is closely related to temperature, pH

value, ion concentration, etc., for example, its value is 224 when Fura-2 is at 37°C; is a

fluorescence intensity ratio of light C when intracellular calcium is zero and saturated; R is a

fluorescence intensity ratio FB/Fc at each measurement point; Rmin is a fluorescence intensity

ratio FB/Fc at zero calcium; Rmax is a fluorescence intensity ratio FB/Fc at saturated calcium,

and the value of Rmax/Rmin is between 13 and 25.

For example, when only light B is used to excite fluorescence, the intensity FB Of

fluorescence D generated by the excitation is detected, and the concentration of free calcium

ions can be calculated through the FB based on the following formula:

[Ca 2 +]j=Kdx(F- Fmn)/(Fmax F)

indicator (such as Fluo-3) and calcium ions, and its value is closely related to temperature, pH

value, ion concentration, etc.; F is a fluorescence intensity FB at each measurement point; Fmin

is a fluorescence intensity FB at zero calcium; and Fmax is a fluorescence intensity FB at

saturated calcium.

As mentioned above, the method includes using light A to be measured to stimulate

fluorescence-stained target cells, using light B and/or light C to perform fluorescence

excitation on the cells, further performing CCD fluorescence imaging on fluorescence D

generated by the excitation, transmitting data to a computer for processing and drawing, and

analyzing real-time changes in free ion concentration to determine the existence of a

photosensitive effect or the response thereof. In the light path of CCD fluorescence imaging, a

filter set for light B and light C is added to eliminate the influence of excitation light B and

light C on the final imaging result; or in the light path of CCD fluorescence imaging, a filter

that allows only light D to pass is added, so that only light D is imaged.

The fluorescent ion indicators of the present invention are a type of chemical substances

that have fluorescence characteristics for specific ions (such as calcium, potassium, and

sodium ions), and the chemical substances cover a great variety of types and have different

chemical principles. In terms of physical characteristics, the absorption wavelength and

emission wavelength of the fluorescent ion indicators are different, that is, light B and/or light

C are determined by the absorption wavelength, and light D is determined by the emitted

fluorescence wavelength; some fluorescent indicators are excited by single-wavelength

monochromatic light, and some fluorescent indicators are excited by dual-wavelength

monochromatic light. Table 1 summarizes recommended wavelengths of light B and light C

when several common fluorescent calcium indicators are used.

Table 1

Indicator Recommended Recommended Kd value name wavelength of light B wavelength of light C

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

Of course, when other specific fluorescent calcium indicators or other fluorescent ion

indicators are used, the light B and/or light C can be determined according to product

instructions or guidelines.

In the method, the wavelengths of light A, light B, light C, and light D are different from

each other; there must be a difference between center values of bands, and the difference is at

least tens of nm or more; the tens of nm is, for example, at least 10 nm, 15 nm, 20 nm, 25 nm,

30 nm, 40 nm, 50 nm or more.

In the method, the light A is a single-band pulsed laser or a combination of light including

pulsed laser measurement sequences of N adjustable parameters of n bands, where n and N are

positive integers, n32, and when N is a combination of measured sequences of laser in n

bands, generally N 3n.

In some embodiments of the present invention, when the light A is a combination of light

including N pulsed laser measurement sequences of n bands, using light A to stimulate target

cells is using pulsed laser of n bands to stimulate the cells respectively in N measurement

sequences.

In the method, the N kinds of pulsed laser of n bands can be freely switched. In some

embodiments of the present invention, when the N kinds of pulsed laser of n bands are freely

switch to stimulate cells respectively, the bands of the pulsed laser for two adjacent

stimulations are different.

In multi-band measurement, multiple parameters of pulsed light can be adjusted

arbitrarily, the parameters including light intensity, pulse width, repetition rate, and timing. The

adjustment of the parameters is usually from small to large (or from weak to strong). In the

measurement of n bands of each group, the parameters should be consistent to ensure the

comparability of the measurement.

In the method, the band center values of the pulsed laser for two adjacent stimulations have a difference of 20-1000 nm, and the difference is preferably 20-800 nm, more preferably

20-400 nm, and most preferably 40 nm.

For example, when light A is a combination of two wavelengths of light or is switched

between two wavelengths of light, that is, n=2, then N>2; the two wavelengths of light are

defined as Ai and A 2 ; when N =2, then when light A is used to stimulate cells, Ai and A 2 are

used to stimulate cells respectively or alternately, indicating that A1 is used to stimulate cells

and then A 2 is used to stimulate the cells, or AIis used to stimulate cells and then switched to

A 2 to stimulate the cells, denoted by Ai--A 2; or A 2 is used to stimulate cells and then A1 is

used to stimulate the cells, or A 2 is used to stimulate cells and then switched to Ai to stimulate

the cells, denoted by A 2-Ai; when N is equal to 3 or greater than 3, using light A to stimulate

cells may be Ai--A 2 -- Ai or A 2-- Ai--A 2 , etc.

In some more specific embodiments, for example, when the present invention is

implemented with the real-time change of calcium ion concentration, the method includes the

following steps:

(1) Cell treatment: selecting target cells for in vitro culture, and adding calcium ions alone

and/or rinsing a culture dish with a solution (such as HBSS solution) that can maintain cell

viability for a short time and contains calcium ions; adding a fluorescent calcium indicator or

fluorescent probes for incubating; selecting suitable cells with fluorescent labels under light B

and light C;

The method for in vitro culture of cells may be a method conventionally mastered by a

person skilled in the art; or the following method for in vitro culture of cells is performed:

selecting cells, performing in vitro culture in a culture dish with a cell slide at a temperature of

37°C in a gas environment of 95% air and 5% C02, adding a suitable cell culture medium to

the culture dish (for example, if nerve cells are cultured, a DMEM-F12 medium special for

nerve cell culture can be used), and culturing for 24 hours.

After the fluorescent indicator or fluorescent probes are added, the incubation time can be

selected according to a conventional method, or the incubation time can be 30-60 minutes.

The fluorescent calcium indicator or fluorescent probes can be, for example, Fura-2, or

other fluorescent calcium indicators as shown in Table 1.

(2) Fluorescent calcium imaging: exciting the fluorescent calcium indicator by light B

and/or light C, and detecting fluorescence intensity FB and/or Fc respectively; calculating an

intensity ratio FB/Fc of fluorescence D on the two excitation wavelengths of light B and light

C, and calculating a concentration of free calcium ions; or calculating a concentration of free

calcium ions by fluorescence intensity FB; wherein, the concentration of calcium ions can be

calculated according to the known formula mentioned above.

(3) Fluorescent calcium imaging under the stimulation of light A: based on the method of

step (2), first fixing a light A emission end to ensure the distance between the light A and the

sample and the incident angle; then, stimulating cells with light A, arranging a filter set that

allow only light D to pass on the imaging light path, performing fluorescent calcium imaging

on light D at the stimulation of light A, and recording changes, wherein when light A is in a

non-visible light band, visible light passes through the same light path and is marked, then the

shooting position of light A is fixed, and the light A is changed to non-visible light to be

measured before fluorescence imaging; finally, calculating a concentration of free calcium ions

under the stimulation of light A.

(4) Drawing a free calcium ion concentration change curve according to the

concentrations of free calcium ions obtained in real time or at regular time in step (2) and step

(3); and comparing changes in the concentrations of free calcium ions on the ion concentration

change curve without stimulation by light A and under stimulation by light A to determine the

existence of a photosensitive effect or further analyze the working status thereof.

The free calcium ion concentration change curve can be drawn directly in step (2) or step

(3) to achieve real-time synchronization. Finally, changes in the concentrations of free calcium

ions on the ion concentration change curve without stimulation by light A and under

stimulation by light A are compared to determine the existence of a photosensitive effect or

further analyze the working status thereof.

In an embodiment of the present invention, the present invention detects auditory nerve

cells, including: using light A to stimulate auditory nerve cells cultured in vitro (for example,

selecting spiral ganglion cells in the cochlear axis), adding a fluorescent calcium indicator if

there are calcium ions in the environment where the cells are located, using light B and light C to perform fluorescence imaging of cells, determining the existence of a photosensitive effect based on the real-time change of free calcium ion concentration or further analyzing the working status thereof. Specifically, the method includes:

(1) Cell treatment: selecting auditory nerve cells (such as spiral ganglion cells) for in vitro

culture, and rinsing a culture dish with HBSS solution (at least once); adding a fluorescent

calcium indicator, such as Fura-2, and incubating for 30-60 min; selecting suitable cells with

fluorescent labels under 340nm ultraviolet light and 380nm ultraviolet light; wherein the

suitable refers to selecting cells with complete morphology and proper position distribution in

a fluorescence-labeled cell image;

(2) Fluorescent calcium imaging: exciting the fluorescent calcium indicator by 340nm

ultraviolet light or 380nm ultraviolet light respectively, detecting fluorescence intensities

respectively, calculating a ratio F 3 40 /F 3 8 0 of the fluorescence intensities under the two

excitation wavelengths, and calculating a concentration of free calcium ions;

wherein the concentration of free calcium ions can be calculated according to a

Grynkiewicz formula.

The Grynkiewicz formula is expressed as follows:

[Ca2+]j=Kdxpx(R - Rmin)/(Rmax - R)

Where, Kd is an equilibrium dissociation constant of binding between Fura-2 and calcium

ions, and its value is closely related to temperature, pH value, ion concentration, etc., and is

224 at 37°C; is a fluorescence intensity ratio at 380 nm when intracellular calcium is zero

and saturated; R is a fluorescence intensity ratio F 34 /F 380 at each measurement point; Rmin is a

fluorescence intensity ratio F 34 /F 38 0 at zero calcium; Rmax is a fluorescence intensity ratio

F 34 /F 38 at saturated calcium, and the value of Rmax/Rmin is between 13 and 25.

step (2), using light A to stimulate cells, performing fluorescent calcium imaging, and

calculating a concentration of free calcium ions under the stimulation of light A; wherein the

light A is a pulsed laser, and its wavelength range may be, for example, any band from 450 nm

to 1065 nm or a combination of any multiple bands;

Using light A to stimulate cells, the light of any wavelength in a wavelength range of 450 nm to 1065 nm (including end values 450 nm and 1065 nm) can be used respectively or in combination to irradiate the cells at different times to provide the stimulation;

When the combined light is used to stimulate alternately, the wavelengths of light for 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; more

accurately, the wavelength difference is a band center value difference.

For example, the cells are stimulated with light of 450 nm or 808 nm or 1065 nm alone; or

the cells are alternately irradiated by a combination of light of at least two wavelengths

selected from the light of 450 nm, 808 nm and 1065 nm, for example, auditory nerve cells are

successively stimulated with 450 nm--808 nm--450 nm pulsed laser (i.e. n=2, N=3);

In one embodiment, when light A is in a non-visible light band, visible light passes

through the same light path or position and is marked, then the shooting position of light A is

fixed, and the light A is changed to non-visible light to be measured before fluorescence

imaging.

(4) Drawing a free calcium ion concentration change curve according to the

existence of a photosensitive effect on the auditory nerve cells or further analyze the working

status thereof.

The method of the present invention for exploring the existence of a photosensitive effect

of the auditory nerve provides an important basis for studying the working mechanism of the

auditory nerve under light stimulation, and is of great significance for further study of

optogenetics in the regulation of the auditory nerve and its application in clinical neurological

diseases.

In the second aspect of the present invention, the present invention provides a system for

real-time screening and measurement of a cellular specific photosensitive effect, including:

a light source A, which emits light A to stimulate target cells;

an ion imaging system, which includes at least a fluorescence excitation light path system and a CCD imaging system, a fluorescence collection light path exciting different fluorescence, and the CCD imaging system detecting fluorescence intensity and collecting image data; and a data processing system, which processes the data collected by the ion imaging system.

Further, in an embodiment of the present invention, the light source A is a multi-band

switchable monochromatic pulsed laser stimulation light source with adjustable parameters, the

parameters including light intensity, pulse width, repetition rate and time.

In an embodiment of the present invention, the light source A can emit visible light and

non-visible light.

In an embodiment of the present invention, the light source A is coupled with an output

light path 1, and the light A can be outputted at different times periods by switching the light

source A and/or adjusting the parameters of the light source A.

In an embodiment of the present invention, the light source A can output single-band

pulsed laser with adjustable parameters or output pulsed laser including n bands in N switching

sequences, where n and N are positive integers, n 2, and N n.

In an embodiment of the present invention, the light source A is a light-emitting device of

general illumination, a light-emitting device of fiber coupled output, or an LED light-emitting

device.

In an embodiment of the present invention, the emission end of the light source A is an

emission port with beam shaping or a natural emission port without beam shaping.

In an embodiment of the present invention, the positions of the light source and the light

path are adjustable, the positions including a distance between the light source or a

long-distance port of the light path away from the light source and a point to be measured or a

region to be measured and an angle of light A irradiated to the point to be measured or the

region to be measured. For example, the positions can be adjusted with a fixing device.

In an embodiment of the present invention, in the ion imaging system, the fluorescence

excitation light path system includes at least two light sources B and C, or at least a light

source capable of implementing switching or coexistence of two light sources B and C;

wherein, the light source B is a characteristic excitation band light source when fluorescent ion probes bind to corresponding ions, and the characteristic excitation light is light

B; the light source C is a characteristic excitation band light source when fluorescent ion

probes do not bind to corresponding ions, and the characteristic excitation light is light C; and

the light source B and the light source C are coupled with an output light path 2 to alternately

output light B and light C to irradiate the target cells and excite fluorescence D.

In an embodiment of the present invention, in the ion imaging system, the CCD imaging

system is provided with an observation light path, a filter is arranged on the observation light

path, and the filter allows only light D to pass or simultaneously block light A, light B and light

C from passing; and the CCD imaging system detects fluorescence through the observation

light path with the filter, collects image data, and transmits the collected data to the data

processing system.

In an embodiment of the present invention, the data collected by the CCD imaging system

is transmitted to the data processing system by a data link, and the data processing system

performs analysis, calculation and drawing on the data to obtain real-time data and images.

Furthermore, in an embodiment of the present invention, the system for real-time

screening and measurement of a cellular specific photosensitive effect according to the present

invention includes:

(1) Light source A (i.e. a light source to be screened), the light source A is a multi-band

switchable pulsed laser stimulation light source (the adjustment or free switching between

different bands can be achieved through, for example, a laser conversion interface or other

components with this function), and the light source A can adjust four parameters including

light intensity, pulse width, repetition rate, and timing;

The light source A is coupled with an output light path 1 (such as a fiber, also referred to

as an output fiber 1 in the present invention, with a diameter of 100 m) through a laser

conversion interface (such as a flange converter) and the light source can be switched to

generate pulsed laser of different bands at different times; the laser outputted from the light

source A is applied to a sample to be measured through the output light path 1 (such as an

output fiber 1, which does not contact the sample); and the light-emitting device of the light

source A may be a fiber-coupled semiconductor laser or LED (such as LED), etc. An emission end (also called an output end) of the light source A is divided into two types, i.e., an emission port with beam shaping and a natural emission port without beam shaping. The light path with beam shaping facilitates better concentration of energy, reduces system power consumption, facilitates the study of precise positioning of the photosensitivity of the target cells, reduces the influence caused by adjusting the position of the light path, and improves detection stability and sensitivity. The light path without beam shaping can increase the irradiation area of stimulation light, increase the range of its photosensitivity, and observe responses of cell photosensitive effects of divergent light at different positions.

The sample to be measured is cells cultured in vitro, and in the environment where the

cells are located, ions to be detected are present and fluorescent ion probes corresponding to

the ions to be detected are added;

The sample to be measured and the output end of light A are fixed by a fixing device, for

example, a dish for accommodating the sample to be measured and a three-dimensional

positioning bracket can be arranged on an operating table of the ion imaging system, and the

three-dimensional positioning bracket can fix the sample to be measured (such as a cell slide)

and the output fiber; the fixing device can also achieve the fixation of the distance between the

light A emission end and the sample to be measured, and the selection of an incident angle of

light A; the output fiber and the sample to be measured should be kept at a proper distance, and

the positions of the cell slide and the output fiber should be always fixed during measurement.

(2) Ion imaging part, including a fluorescent probe excitation light path system and a

CCD imaging system;

1) Fluorescent probe excitation light path system, the light path includes at least two light

sources B and C or can implement the switching and coexistence of two light sources B and C,

and the light source B and/or light source C are characteristic excitation band light sources for

a fluorescent ion indicator; the light source B or the light source C irradiates the sample to be

measured or the light source B and the light source C alternately irradiate the sample to be

measured (the light irradiates the sample to be measured through an output light path 2, such as

an output fiber 2), to excite different intensities of fluorescence, and the fluorescence produced

by the excitation is fluorescence D;

2) CCD imaging system, which detects fluorescence intensity and collects image data

through an observation light path (such as a CCD), and transmits the data to a data processing

system through a data link; the observation light path should include a set of broadband

adjustable band filters, which can be filters for light A, light B and light C, or a filter that can

transmit only fluorescence D, thereby eliminating the interference of light A, B, and C, and

allowing the collected fluorescence D to undergo CCD imaging;

In addition, the observation light path further contains an observation light path branch

leading to an optical microscope. Cells in the field of view and the position of the output fiber

are observed through an observation eyepiece of the optical microscope, so as to ensure that

the laser to be measured can accurately irradiate the target cells through the output light path 1

(output fiber 1). The observation optical branch and the optical microscope connected thereto

can be removed or closed after determining the cells and the position of the output fiber. They

do not directly participate in the real-time screening and measurement of the photosensitive

effect.

Or other devices integrated with the above functions are used to realize the detection of

fluorescence intensity and the synchronous collection of image data;

(3) Data processing system, which performs calculation and drawing on the data collected

and synchronously transmitted by the ion imaging system to obtain a corresponding real-time

ion concentration curve;

The data processing system (such as a data processing computer) can use, for example,

MetaFluor fluorescence ratio imaging software, which can simultaneously display original

data, ratio images, fluorescence intensity curve charts, ratio curve charts, ion concentration

curve charts, and non-proportional measured images such as bright fields or phase difference

imaging. The software can simultaneously image and measure two different ratio measurement

indicators without being affected by dye loading concentration, condition or emission intensity.

In addition, it should be noted that both light output and input require light paths (such as

fibers, beam shaping, CCD), and the connection between the light paths and corresponding

devices or components can be implemented by circuit interfaces or conversion interfaces that

are well-known in the art.

In one embodiment of the present invention, the system of the present invention is shown

in Fig. 2.

In the third aspect of the present invention, the present invention provides a method for

real-time screening and measurement of a photosensitive effect of cells using the system shown

above, the method including the following steps:

(1) Cell treatment: selecting target cells for in vitro culture, and adding ions to be detected

alone or rinsing a culture dish with a solution that can maintain cell viability for a short time

and contains ions to be detected; adding a fluorescent ion indicator corresponding to the ions to

be detected and then incubating; selecting suitable cells with fluorescent labels under the

excitation of a light source B and/or a light source C;

After the cell treatment is completed, taking a cell slide from the culture dish, putting the

cell slide into a small dish of an operating table of an ion imaging system, and fixing the

positions of the cell slide and a light A output port by adjusting a three-dimensional positioning

bracket, wherein the light A output port should be kept at a proper distance from the cells and

should not be in contact with the cells, and in order to avoid fluorescence quenching, the whole

process should be carried out under dark light conditions; Observing the cells in the field of

view and the position of the light A output port through an optical microscope; Closing the light of

the optical microscope, performing fluorescence labeling with laser (excited by fluorescent probes)

of a specific wavelength corresponding to a specific fluorescent ion indicator, and selecting cells

with complete morphology and proper position distribution from the fluorescence-labeled cell

image;

(2) Fluorescent ion imaging: exciting the light source B and/or the light source C of a

light path system respectively by fluorescent probes to excite the fluorescent ion indicator,

detecting, by a CCD imaging system, resting intensity values of fluorescence (i.e. fluorescence

D as described above) emitted by free ions and bound ion in the fluorescent probes and the

target cells respectively, transmitting to a data processing system in real time, and calculating a

real-time or timed concentration of free ions to be detected without the stimulation of a light

source A;

(3) Fluorescent ion imaging under the stimulation of the light source A: turning on the light source A to stimulate cells, repeating the operation of step (2), performing fluorescent ion imaging under the stimulation of the light source A, and calculating a real-time or timed concentration of free ions under the stimulation of the light source A;

(4) The CCD imaging system detecting fluorescence intensities in steps (2) and (3) and

transmitting data to the data processing system; the data processing system calculating the data

and drawing a real-time ion concentration curve; and

(5) Comparing changes in the concentrations of free ions on the real-time ion

concentration change curve without stimulation by light A and under stimulation by light A to

determine the existence of a photosensitive effect or further analyze the working status thereof.

In the above method, the light source A generates only single-band pulsed laser with

specific parameters at the same time, and the light source A can switch bands at different times

and adjust parameters at any time, the parameters including light intensity, pulse width,

repetition rate, and time;

In the detection process, the light source A can generate a combination of N pulsed laser

measurement sequences of n bands, where n and N are positive integers, n 2, and N n; the

N pulsed laser measurement sequences of n bands can be freely switched;

Preferably, when N kinds of pulsed laser of n bands are freely switched to stimulate cells

at different times, the bands of pulsed laser for two adjacent stimulations are different;

Preferably, the bands of the pulsed laser for two adjacent stimulations have a difference of

20-1000 nm, and the difference is preferably 20-800 nm, more preferably 20-400 nm, and most

preferably 40 nm.

Preferably, the wavelengths of light A, light B, light D, and light C are different from each

other. In some embodiments of the present invention, the wavelengths (band center values) of

light A, light B, light D, and light C have differences of at least tens of nm or more, such as 10

nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm or 50 nm (determined according to the

monochromaticity of light source devices).

Preferably, the ions to be detected are, for example, calcium ions, and the fluorescent ion

indicator is, for example, selected from any one in Table 1.

In another aspect of the present invention, the present invention provides a method for real-time screening and measurement of a cellular specific photosensitive effect, the method comprising: using light A to be measured to stimulate target cells, wherein the target cells being living cells in vitro and fluorescently stained using a fluorescent ion indicator; using light B and/or light C which are excitation light for the fluorescent ion indicator to perform fluorescence excitation on the target cells and to generate fluorescence D, detecting fluorescence intensity through ion imaging technology, and performing a data processing, then drawing a specific curve of real-time or timed ion concentration changes to determine the existence of the cellular specific photosensitive effect or further analyze the working status thereof; wherein, the light A to be measured is a monochromatic pulsed laser with adjustable parameters, the parameters comprise wavelength, light intensity, pulse width, repetition rate, and time, and the light A is visible light or invisible light; the light A is a single-wavelength pulsed laser or a combination of N pulsed laser measurement sequences including n bands, wherein n and N are positive integers, n32, and N

2n;

when the light A is a combination of N pulsed laser measurement sequences including n

bands, using light A to stimulate target cells is using pulsed laser of n bands to stimulate the

target cells respectively in N measurement sequences;

the N kinds of pulsed laser of the n bands can be freely switched;

when the N kinds of pulsed laser of the n bands are freely switched to stimulate cells at

different times, the bands of pulsed laser for two adjacent stimulations are different; and

band center values of the pulsed laser for two adjacent stimulations have a difference of

20-1000 nm;

wherein, when light A is in a non-visible light band, visible light passes through the same

light path or position and is marked, then the shooting position of light A is fixed, and the light

A is changed to non-visible light to be measured before fluorescence imaging at the stimulation

of light A;

wherein, the wavelengths of light A, light B, light C and light D are different from each

other; and the wavelength differences between the light A, the light B, the light C, and the light D are greater than 10 nm.

In another aspect of the present invention, the present invention provides a system for

real-time screening and measurement of a cellular specific photosensitive effect, the system

comprising: a light source A, which emits light A to stimulate target cells;

an ion imaging system, which includes at least a fluorescence excitation light path system

and a CCD imaging system that collects emitted fluorescence,

a fluorescence excitation light path exciting fluorescence of different ions, and the CCD

imaging system detecting fluorescence intensity and collecting image data; and

a data processing system, which processes the data collected by the ion imaging system;

wherein the light source A is a multi-band switchable monochromatic pulsed laser

stimulation light source with adjustable parameters, the parameters comprising light intensity,

pulse width, repetition rate and time;

the light source A can emit visible light and non-visible light;

the light A is coupled with an output light path 1, and the light A can be outputted at

different times periods by switching bands (1, 2, 3...n) of light sources and/or adjusting the

parameters of the light source A;

the light source A can output single-band pulsed laser with adjustable parameters or

output pulsed laser including n bands in a combination of N switching sequences, where n and

N are positive integers, n:2, and N:n;

wherein, the fluorescence excitation light path system comprises at least two light sources

B and/or C, or at least a light source capable of implementing switching or coexistence of two

light sources B and C;

the light source B is a characteristic excitation band light source when fluorescent probes

bind to corresponding ions, and the characteristic excitation light is light B; the light source C

is a characteristic excitation band light source when fluorescent ion probes do not bind to

corresponding ions, and the characteristic excitation light is light C; the light source B and the

light source C are coupled with an output light path 2 to alternately output light B and light C

to irradiate the target cells and excite fluorescence D; wherein, the CCD imaging system is provided with an observation light path, a filter is arranged on the observation light path, and the filter allows only light D to pass or simultaneously block light A, light B and light C from passing; and the CCD imaging system detects fluorescence through the observation light path with the filter, collects image data, and transmits the collected data to the data processing system; and the data collected by the CCD imaging system is transmitted to the data processing system by a data link, and the data processing system performs analysis, calculation and drawing on the data to obtain real-time data and images.

In another aspect of the present invention, the present invention provides a method for

real-time screening and measurement of a cellular specific photosensitive effect, the method

based on the system of any one of claims 8 to 12, comprising the following steps:(1) cell

treatment: selecting target cells for in vitro culture, and adding ions to be detected alone or

rinsing a culture dish with a solution that can maintain cell viability for a short time and

contains ions to be detected; adding a fluorescent ion indicator for the ions to be detected and

then incubating; selecting suitable cells with fluorescent labels under a light source B and/or a

light source C;(2) fluorescent ion imaging: exciting the fluorescent ion indicator by light B

and/or light C, the CCD imaging system synchronously detecting fluorescence and collecting

image information, and transmitting the information to the data processing system in real

time;(3) fluorescent ion imaging under the stimulation of light A: turning on a light source A to

stimulate the cells, repeating the operation of step (2), performing intracellular fluorescent ion

imaging under the stimulation of light A, the CCD imaging system synchronously detecting

fluorescence and collecting image information, and transmitting the information to the data

processing system in real time,

when light A is in a non-visible light band, visible light passes through the same light path

or position and is marked, then the shooting position of light A is fixed, and the light A is

changed to non-visible light to be measured before fluorescence imaging;(4) the data

processing system analyzing and calculating the information of step (2) and step (3) received

in real time, and drawing a real-time ion concentration curve chart; and(5) comparing changes

in the concentrations of ions on the real-time ion concentration change curve without stimulation by light A and under stimulation by light A to determine the existence of a photosensitive effect or further analyze the working status thereof;wherein, the light source A can switch bands at different times and adjust parameters at any time, the parameters comprise light intensity, pulse width, repetition rate and time, and the light source A generates only single-band pulsed light with specific parameters at the same time; in the detection process, the light source A can generate a combination of N pulsed laser measurement sequences of n bands, where n and N are positive integers, n >2, and N>n; the

N kinds of pulsed laser of n bands can be freely switched;

when the N kinds of pulsed laser of n bands are freely switched to stimulate cells at

different times, the bands of pulsed laser for two adjacent stimulations are different;

20-1000 nm;

the band center values of light A, light B, light C, and light D are different from each

other; and

the band center value difference between the light A, light B, light C, and light D is at

least 10 nm or more.

Brief Description of the Drawings

Hereinafter, the implementation schemes of the present invention will be described in

detail with reference to the accompanying drawings, in which:

Fig. 1 is a schematic principle diagram of a method according to the present invention;

wherein (a) shows ions inside and outside a cell that has not been irradiated by light of a

specific wavelength; (b) shows ions inside and outside a cell that has been irradiated by light of

a specific wavelength.

Fig. 2 is a schematic diagram of a system for real-time screening and measurement of a

cellular specific photosensitive effect according to the present invention. The system contains 3

light sources, wherein the light source A (that is, a light source to be measured) is pulsed laser

with adjustable parameters such as light intensity, pulse width, repetition rate, and time in

bands to be screened. The light source emits pulsed laser with corresponding parameters from a parameter-adjustable laser or a light-emitting device. The generated pulsed optical signals of specific parameters are coupled with an output light path 1 through a light conversion interface and the light source is switched to generate pulsed light of different bands in different time periods. The optical signals are irradiated to corresponding sample target cells through the output light path 1. The light source B and the light source C are two light sources in a fluorescent probe excitation light path system, which are respectively characteristic excitation light sources when ions bind to and do not bind to probes. The light source B and the light source C alternately irradiate the sample target cells through an output light path 2, the stained samples to be measured are excited to generate corresponding fluorescence D, image data is collected by a CCD imaging system and transmitted to a data processing computer by a data link, data calculation and drawing are performed by image processing software, and a corresponding real-time ion concentration curve is obtained.

Fig. 3 shows a field of view of a nerve cell to be measured and a light output port to be

measured under an optical microscope.

Fig. 4 shows the selection of auditory nerve target cells in the imaging field of view after

fluorescent labeling.

Fig. 5 shows a process of Fura-2 binding to calcium ions and a schematic spectrogram of

excitation light and emission light.

Fig. 6 shows measurement results of auditory nerve cells without stimulation by external laser

(light A), Fig. 6A shows 6 selected target cells, numbered by 1, 2, 3, 4, 5, and 6, and Fig. 6B shows

real-time ion concentration curves corresponding to the selected cells.

Fig. 7 shows measurement results of auditory nerve cells under stimulation by 450 nm pulsed

laser (light A), Fig. 7A shows 6 selected target cells, numbered by 1, 2, 3, 4, 5, and 6, and Fig. 7B

shows real-time ion concentration curves corresponding to the selected cells.

Fig. 8 shows measurement results of sequentially stimulating auditory nerve cells by pulsed

laser with wavelengths 450 nm-808 nm--450 nm, Fig. 8A shows 6 selected target cells,

numbered by 1, 2, 3, 4, 5, and 6, and Fig. 8B shows real-time ion concentration curves

corresponding to the selected cells.

Detailed Description of the Embodiments

The present invention will be further explained below in conjunction with specific

embodiments. It should be understood that these embodiments are only used to illustrate the

present invention and not to limit the scope of the present invention. The experimental methods

that do not indicate specific conditions in the following embodiments are usually in accordance

with conventional conditions or in accordance with the conditions recommended by the

manufacturer.

Unless otherwise defined, all professional and scientific terms used here have the same

meanings as those familiar to those skilled in the art. In addition, any method and material

similar or equivalent to the content described can be applied to the method of the present

invention. The preferred implementation methods and materials described here are for

demonstration purposes only.

The following embodiments are examples of the measurement method of the present

invention. In the following embodiments, it was found by measuring auditory nerve cells with

the method of the present invention that there is a photosensitive effect among the auditory

nerve cells, and the photosensitive effect is selective for the wavelength of irradiation light. It

should be understood that the method of the present invention is not specifically limited to the

auditory nerve cells used in the following embodiments, and the cells may be any cells desired

to be measured. Moreover, light A in the present invention, as pulsed light to be measured, is a

fast-switching monochromatic light source whose wavelength, light intensity, pulse width,

repetition rate, and time can be adjusted.

The general process of this embodiment is as follows: spiral ganglion cells in the cochlear

axis of a 7-days C57-BL young black rat provided by the Animal Experiment Center of Shandong

University are selected, and cultured in vitro in a culture dish with a cell slide, the temperature of

the culture dish is controlled at 37C, the gaseous environment contains 95% air and 5% C02, a

DMEM-F12 medium special for nerve cell culture is added, and the cells are cultured in dark for

24 hours. After that, the medium is removed, and the culture dish is rinsed twice with an HBSS

solution that can maintain cell viability for a short time, the buffer solution including a large

amount of Ca2. Next, a specific fluorescent calcium indicator is added and the cells are incubated for 30-60 minutes. After the pretreatment, the cell slide is taken out from the culture dish and placed in a small dish on an operating table of a calcium imaging system, a three-dimensional positioning bracket is adjusted, and under aided observation in the field of view of an optical microscope, the positions of the cell slide and an output fiber are fixed. The fiber should be kept at a proper distance from the cells, and cannot contact the cells. The fiber used here has a diameter of

100 m. In order to avoid fluorescence quenching, the whole process should be carried out under

dark light conditions. The cells and the position of the fiber in the field of view are observed

through the optical microscope, as shown in Fig. 3. The optical microscope is turned off,

fluorescence labeling is performed under the excitation of laser of a specific wavelength

corresponding to a specific fluorescent calcium indicator, and cells with complete morphology and

proper position distribution are selected from the fluorescence-labeled cell image, as shown in Fig.

4. The four parameters of laser outputted by a light source to be measured (i.e., light A), i.e., light

intensity, pulse width, repetition rate, and time, can be adjusted arbitrarily. In order to avoid

deactivation of the target cells, it is recommended that the laser application time is reasonably

selected according to the energy of the outputted laser. Multiple light sources to be measured with

different wavelengths can be freely switched by a laser conversion interface, and the laser is

applied to the target cells by the fiber to screen for a photosensitive effect. After the parameters of

the light source A to be measured are set, excitation light B and C are selected. At this time,

fluorescent probe excitation starts to operate. The wavelengths of outputted light (i.e. excitation

light B and/or C) can be switched for the absorption wavelengths of different specific fluorescent

calcium indicators or light of multiple wavelengths is outputted at the same time. Meanwhile, the

light source A is turned off or on, and Meta Flour analysis software draws a real-time fluorescence

D intensity curve to reflect the change in the concentration of calcium ions in the selected cells.

Based on the above operation, when the light source A is turned off, the curve reflects the real-time

calcium ion concentration of the cells in a resting state or without external light stimulation, and

when the light source A is turned on, the curve reflects the real-time calcium ion concentration of

the cells under the stimulation of external light. In order to avoid unnecessary interference, the

wavelength of the light source to be measured (i.e. light A) should be kept at a difference of

several tens of nm from the absorption wavelength of the specific fluorescent calcium indicator

(i.e. excitation light B or C) and the emission wavelength of fluorescence (i.e. light D). For

example, in order to ensure the accuracy of the experiment, the difference should be kept above 40

nm in this embodiment. During the experiment, the positions of the cell slide and the output fiber

should be always fixed.

Embodiment

1. Cell treatment

Spiral ganglion cells in the cochlear axis of a 7-days C57-BL young black rat provided by the

Animal Experiment Center of Shandong University were selected, and cultured in vitro in a culture

dish with a cell slide, the temperature of the culture dish was controlled at 37°C, the gaseous

environment contained 95% air and 5% C02, a DMEM-F12 medium special for nerve cell culture

was added, and the cells were cultured in dark for 24 hours. After that, the medium was removed,

and the culture dish was rinsed twice with an HBSS solution that can maintain cell viability for a

short time, the buffer solution including a large amount of Ca2. Next, a specific fluorescent

calcium indicator Fura-2 was added and the cells were incubated for 30-60 minutes. After the

pretreatment, the cell slide was taken out from the culture dish and placed in a small dish on an

operating table of a calcium imaging system, a three-dimensional positioning bracket was

adjusted, and the positions of the cell slide and an output fiber were fixed. The fiber should be kept

at a proper distance and angle from the cells, and cannot contact the cells. The fiber used here had

a diameter of 100 m. In order to avoid fluorescence quenching, the whole process should be

carried out under dark light conditions. The cells and the position of the fiber in the field of view

were observed through the optical microscope, as shown in Fig. 3. The optical microscope was

turned off, fluorescence labeling was performed by laser with absorption wavelengths of 340 nm

and 380 nm corresponding to Fura-2, and cells with complete morphology and proper position

distribution were selected from the fluorescence-labeled cell image, as shown in Fig. 4. Then the

follow-up test experiment was carried out. 2. Fluorescent calcium imaging

The working principle of a fluorescent calcium indicator: Fura-2 was currently the most

commonly used fluorescent calcium indicator (also called fluorescent calcium probe), was a

chemical calcium indicator among the indicator types, and can specifically bind to intracellular free calcium ions. Fura-2 was excited by ultraviolet light, the excitation wavelength of the bound

Fura-2 was 340 nm, the excitation wavelength of the free Fura-2 was 380 nm, as shown in Fig. 5,

and their emission spectrum peaks were at 505 to 520 nm without significant changes. Therefore,

the ratio of the calcium-bound Fura-2 to the unbound Fura-2 can be determined by detecting the

ratio F 3 4 /F 3 8 0 of fluorescence intensities on the two excitation wavelengths, and then the

concentration of free calcium ions can be solved based on the Grynkiewicz formula. The

Grynkiewicz formula was expressed as follows:

[Ca2+]j=Kdxpx(R - Rmin)/(Rmax - R)

Where, Kd was an equilibrium dissociation constant of binding between Fura-2 and calcium

ions, and its value was closely related to temperature, pH value, ion concentration, etc., and was

224 at 37°C; was a fluorescence intensity ratio at 380 nm when intracellular calcium was zero

and saturated; R was a fluorescence intensity ratio F 34 /F 380 at each measurement point; Rmin was a

fluorescence intensity ratio F 34 /F 38 0 at zero calcium; Rmax was a fluorescence intensity ratio

F 34 /F 38 at saturated calcium, and the value of Rmax/Rminwas between 13 and 25.

Calcium imaging system: after the cell treatment was completed, the intracellular Ca2+

concentration of the selected cells was measured using the calcium imaging system. Before the

measurement, the fiber of the light A to be measured was fixed with a three-dimensional regulator

(i.e. the aforementioned three-dimensional positioning bracket) to ensure the distance and angle

between the light A emission end and the sample in the experiment. During the experiment, the

positions of the cell slide and the output fiber should be always fixed.

Before the light to be measured was applied, the four parameters of light sources to be

measured with different wavelengths, i.e., light intensity, pulse width, repetition rate, and time,

should be set. When light A to be measured was not applied, a fluorescence intensity in a resting

state was first collected using a fluorescent probe excitation light path system for resting cells, the

wavelength of outputted light was switched between the two absorption wavelengths 340 nm and

380 nm of Fura-2, and a real-time fluorescence intensity curve was drawn by data analysis

software to reflect the resting status of the calcium ion concentration of the selected cells. When

the auditory nerve cells were irradiated by light A, the light sources to be measured with three

different wavelengths of 450 nm, 808 nm and 1065 nm were freely switched by a laser conversion interface, and the laser was applied to the target cells by the fiber to screen and measure a photosensitive effect. 3. Fluorescent calcium imaging under the stimulation of light A

Measurement results without external laser stimulation (i.e. the stimulation of light A was not applied)

When there was no external laser signal irradiation, the calcium ion concentration of the cell

sample was collected (2 ms/time). As shown in Fig. 6, the intracellular Ca2+ concentrations of the

six nerve cells selected under the microscope imaging field do not change significantly. It can be

considered that the auditory nerve cells were in a resting state at this time. The test results can be

used as a reference for cell response under the stimulation of external laser later.

Single-wavelength laser stimulation experiment of auditory nerve cells

Considering the tolerance of animal cells to energy laser, and avoiding long-time

high-intensity laser stimulation that may inactivate the cells, the experiment used grouped

intermittent irradiation of laser signals, and each group of cells was only irradiated by a single

wavelength of laser. In order to ensure cell viability, after a test was completed, another group of

new cells was used for subsequent experiments. The target cells should be cultured in a suitable

dark environment to avoid fluorescence quenching of cells. For the three single-wavelength laser

stimulations, the observations of changes in the calcium ion concentration of nerve cells were as

follows.

(1) Measurement results of 450nm pulsed laser stimulation

450 nm pulsed laser was applied, repetition rate 11 Hz, pulse width 300 [s, light intensity:

gradually increase from zero, calcium ion data collection speed: 2 ms/time (collection rate: 500

times/sec).

External optical signals (corresponding to the white line at the bottom of Fig. 7B) were

started. During continuous irradiation, significant changes in the intracellular Ca2+ concentration

were measured, as shown in Fig. 7. It can be discovered by careful observation that the response

peak of the Ca2+ concentration tends to gradually rise over time, until a maximum critical point,

the peak of the Ca2+ concentration gradually decreased.

This phenomenon showed that the external pulsed laser signals with a wavelength of 450 nm caused nerve impulses in animal auditory nerve cells, which induced the transduction function of the auditory nerve. Observing the positions of the cells and the fiber again, it can be found that the fiber in this experiment was placed at the upper right of thefield of the microscope. Among the six cells selected in the field of the microscope, the nerve impulses generated by the cells (No. 2, 3, 5,

6) closer to the fiber port were more obvious, while the amplitude of each impulse generated by

the nerve cells (No. 1, 4) far away from the fiber was smaller, although the times of generating

nerve impulses were substantially consistent. This phenomenon may have certain relationship with

the energy of an output light spot at the fiber port, because the light spot was a circular surface

with a diameter of 100 m, and its illumination or energy gradually attenuated from the center to

four sides.

(2) 808nm pulsed laser stimulation results

Another group of cells was used, the same parameters were set, and the cells were irradiated

by 808nm laser. During the same period of laser irradiation, the intracellular Ca2+ concentrations

of the six nerve cells selected in the field of the microscope did not change significantly (as shown

in Fig. 6A). Although the Ca2+ concentration in one or two cells showed a slight rising trend, it was

found by quantifying the values of vertical coordinates that the ratio of the concentration

increment to the total concentration was less than 3%o, which can almost be considered that the

auditory nerve cells did not produce any impulse. Experiments showed that the pulsed laser with a

wavelength of 808 nm had no significant influence on the transduction of nerve cells.

(3) 1065nm pulsed laser stimulation results

Same as above, after 1065nm laser irradiated cells, the intracellular Ca2+ concentrations of the

six nerve cells selected in the field of the microscope did not change significantly before and after

laser irradiation, which can almost be considered that the auditory nerve cells did not produce

corresponding impulses. Experiments showed that the pulsed laser with a wavelength of 1065 nm

had no significant influence on the transduction of nerve cells.

Stimulation experiments by switching laser of different wavelengths

In the single-wavelength laser stimulation test on auditory nerve cells, a different group was

tested each time. In order to eliminate the possible differences in cell activity of different groups of

cells, the same group of cells was successively irradiated by laser signals of different wavelengths to further explore the selective response of animal auditory nerve cells to laser wavelengths.

In this experiment, two wavelengths 450 nm and 808 nm of laser were switched as 450

nm--808 nm--+450 nm. The measurement results were shown in Fig. 8. It can be found that when

the cells were irradiated by 450nm laser (the time axis corresponded to a first small white column,

starting from the left on the abscissa axis), the intracellular Ca2+ concentrations of the selected six

nerve cells changed significantly; then the laser signal was turned off, the wavelength parameter of

the light source was changed to 808 nm, other parameters remained unchanged, the cells were

irradiated again (the time axis corresponded to a second small white column, starting from the left

on the abscissa axis), and it can be found that the intracellular Ca2+ concentrations did not have

obvious changes during a long time of irradiation; later, the 808 nm laser was turned off, the

wavelength of laser was adjusted back to 450nm for irradiation (the time axis corresponded to a

third small white column, starting from the left on the abscissa axis), it can be found that the

intracellular Ca2+ concentration of only one cell among the selected six cells increased

significantly, but the amplitude of impulse generated was significantly weaker than that of the

calcium ion concentration response of the cell when irradiated by the 450 nm laser before. This

phenomenon may be related to the fact that after a period of 808 nm laser irradiation, although

there was no significant change in calcium ions, the activity of the nerve cells may be affected to a

certain extent, and after the nerve cells were irradiated by the 450 nm light again, the transduction

response of the cells decreased.

Experimental results: three kinds of pulsed laser with different wavelengths 450 nm, 808

nm and 1065 nm were selected to stimulate animal auditory nerve cells, the results confirmed that

the 450 nm laser can significantly change the intracellular Ca2+ concentration, and it was found

that the position of the fiber output port was related to the stimulation response of auditory cells;

and the stimulation by laser of other two wavelengths (808 nm and 1065 nm) did not significantly

change the intracellular Ca2+ concentration of the auditory nerve cells. This preliminarily

confirmed that the auditory cells can produce nerve impulses under the irradiation of laser signals

of suitable wavelengths, and there was a phenomenon of cation transduction, or, the laser-induced

auditory nerve response had wavelength selectivity.

Furthermore, different wavelengths (450 nm and 808 nm) of laser were switched to stimulate the auditory nerve cells, the results confirmed that the auditory nerve cells were indeed sensitive to the 450 nm light, the 450 nm light can repeatedly trigger significant changes in calcium ion concentration and trigger nerve impulses or transduction responses, and the cells did not have obvious nerve impulse response under the stimulation of laser of non-sensitive bands.

Experiments have preliminarily confirmed, the light stimulation that can induce changes in

the calcium ion concentration of isolated auditory nerve cells to trigger nerve transduction had

wavelength selectivity, and 450nm was its sensitive band, which was easier to stimulate the release

of transmitters required for the nerve transduction. This transduction phenomenon, inferred from

the perspective of optogenetics, should have a photosensitive effect corresponding to 450 nm on

auditory nerve cells.

Using calcium imaging technology to measure obvious changes in the intracellular Ca2+

concentration caused by the laser of 450 nm band, it can be concluded that there was a relevant

photosensitive effect on the auditory nerve cell membrane, and a large amount of influx of Ca2+ n

the extracellular fluid led to a sharp rise in the intracellular Ca2+ concentration; and after the 450

nm laser was removed, the intracellular Ca2+ concentration gradually decreased. This phenomenon

was consistent with the general phenomenon of cell electrophysiological measurement.

In addition, although the 340 nm/380 nm collection light band and the fluorescence emission

light bands (505 nm to 520 nm) used when the calcium imaging system collected Ca2+

concentration data were close to the 450 nm band of the laser to be measured, there was still a

distance of more than 50 nm, which can be considered that there was no obvious interference of

optical signals.

Claims

1. A method for real-time screening and measurement of a cellular specific photosensitive

effect, the method comprising: using light A to be measured to stimulate target cells, wherein

the target cells being living cells in vitro and fluorescently stained using a fluorescent ion

indicator; using light B and/or light C which are excitation light for the fluorescent ion

indicator to perform fluorescence excitation on the target cells and to generate fluorescence D,

detecting fluorescence intensity through ion imaging technology, and performing a data

processing, then drawing a specific curve of real-time or timed ion concentration changes to

determine the existence of the cellular specific photosensitive effect or further analyze the

working status thereof;

wherein, the light A to be measured is a monochromatic pulsed laser with adjustable

parameters, the parameters comprise wavelength, light intensity, pulse width, repetition rate,

and time, and the light A is visible light or invisible light;

the light A is a single-wavelength pulsed laser or a combination of N pulsed laser

measurement sequences including n bands, wherein n and N are positive integers, n>2, and

N>n;

target cells respectively in N measurement sequences;

the N kinds of pulsed laser of the n bands can be freely switched;

20-1000 nm;

of light A;

wherein, the wavelengths of light A, light B, light C and light D are different from each other; and the wavelength differences between the light A, the light B, the light C, and the light D are greater than 10 nm.

2. The method according to claim 1, wherein the band center values of the pulsed laser for

two adjacent stimulations have a difference of 20-800 nm.

3. The method according to claim 1, wherein the band center values of the pulsed laser for

two adjacent stimulations have a difference of 20-400 nm.

4. The method according to claim 1, wherein the light A has an optional wavelength range

of 450 nm to 1065 nm.

5. The method according to claim 1, wherein the light A is a combination of N pulsed

laser measurement sequences including n bands, the combination being selected from a

combination of light of at least two wavelengths selected from 450 nm, 808 nm and 1065 nm.

6. The method according to claim 1, wherein the target cells are auditory nerve cells, and

the light A is a combination of light of wavelengths 450 nm--808 nm--450 nm.

7. The method according to any one of claims 1 to 6, wherein the method comprises:

be detected and then incubating; selecting suitable cells with fluorescent labels under light B

and/or light C;

(2) fluorescent ion imaging: exciting the fluorescent ion indicator by light B and/or light

C, and detecting intensity FB and/or Fc of fluorescence D respectively; calculating an intensity

ratio FB/Fc of fluorescence D on the two excitation wavelengths of light B and light C, and

calculating a concentration of intracellular ions; or calculating a concentration of intracellular

ions by the intensity FBof fluorescence D;

(3) fluorescent ion imaging under the stimulation of light A: according to the method in

step (2), using light A to stimulate cells, and performing fluorescent ion imaging under the

stimulation of light A; calculating changes in the concentration of intracellular ions under the

stimulation of light A; and

(4) drawing an ion concentration change curve according to the concentrations of ions

obtained in real time or at regular time in step (2) and step (3); and comparing changes in the

concentrations of ions on the ion concentration change curve without stimulation by light A

and under stimulation by light A to determine the existence of a photosensitive effect of the

light A to be measured or further analyze the working status thereof.

8. A system for real-time screening and measurement of a cellular specific photosensitive

effect, the system comprising:

a light source A, which emits light A to stimulate target cells;

and a CCD imaging system that collects emitted fluorescence,

imaging system detecting fluorescence intensity and collecting image data; and

pulse width, repetition rate and time;

the light source A can emit visible light and non-visible light;

parameters of the light source A;

N are positive integers, n 2, and N>n;

light sources B and C;

is a characteristic excitation band light source when fluorescent ion probes do not bind to corresponding ions, and the characteristic excitation light is light C; the light source B and the light source C are coupled with an output light path 2 to alternately output light B and light C to irradiate the target cells and excite fluorescence D; wherein, the CCD imaging system is provided with an observation light path, a filter is arranged on the observation light path, and the filter allows only light D to pass or simultaneously block light A, light B and light C from passing; and the CCD imaging system detects fluorescence through the observation light path with the filter, collects image data, and transmits the collected data to the data processing system; and the data collected by the CCD imaging system is transmitted to the data processing system by a data link, and the data processing system performs analysis, calculation and drawing on the data to obtain real-time data and images.

9. The system according to claim 8, wherein the light source A is a light-emitting device

of general illumination, a light-emitting device of fiber coupled output, 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 positions of the light source and the light path are adjustable, the positions comprising

a distance between the light source or a long-distance port of the light path away from the light

source and a point to be measured or a region to be measured and an angle of light A irradiated

to the point to be measured or the region to be measured.

10. The system according to claim 8, wherein the light A has an optional wavelength

range of 450 nm to 1065 nm.

11. The system according to claim 8, wherein the light A is a combination of N pulsed

12. The system according to claim 8, wherein the target cells are auditory nerve cells, and

the light A is a combination of light of wavelengths 450 nm--808 nm--450 nm.

13. A method for real-time screening and measurement of a cellular specific

photosensitive effect, the method based on the system of any one of claims 8 to 12, comprising the following steps:

and contains ions to be detected; adding a fluorescent ion indicator for the ions to be detected

and then incubating; selecting suitable cells with fluorescent labels under a light source B

and/or a light source C;

C, the CCD imaging system synchronously detecting fluorescence and collecting image

information, and transmitting the information to the data processing system in real time;

(3) fluorescent ion imaging under the stimulation of light A: turning on a light source A to

processing system in real time,

changed to non-visible light to be measured before fluorescence imaging;

(4) the data processing system analyzing and calculating the information of step (2) and

step (3) received in real time, and drawing a real-time ion concentration curve chart; and

(5) comparing changes in the concentrations of ions on the real-time ion concentration

existence of a photosensitive effect or further analyze the working status thereof;

wherein, the light source A can switch bands at different times and adjust parameters at

any time, the parameters comprise light intensity, pulse width, repetition rate and time, and the

light source A generates only single-band pulsed light with specific parameters at the same

time;

measurement sequences of n bands, where n and N are positive integers, n 2, and N:n; the

N kinds of pulsed laser of n bands can be freely switched; when the N kinds of pulsed laser of n bands are freely switched to stimulate cells at different times, the bands of pulsed laser for two adjacent stimulations are different; band center values of the pulsed laser for two adjacent stimulations have a difference of

20-1000 nm;

other; and

least 10 nm or more.

14. The method according to claim 13, wherein the band center values of the pulsed laser

for two adjacent stimulations have a difference of 20-800 nm.

15. The method according to claim 13, wherein the band center values of the pulsed laser

for two adjacent stimulations have a difference of 20-400 nm.

16. The method according to claim 13, wherein the light A has an optional wavelength

range of 450 nm to 1065 nm.

17. The method according to claim 13, wherein the light A is a combination of N pulsed

18. The method according to claim 13, wherein the target cells are auditory nerve cells,

and the light A is a combination of light of wavelengths 450 nm--808 nm--450 nm.