Light source device and method for applying light source device to optogenetics tool protein excitation spectrum measurement

Abstract

The invention discloses a light source device and a method for applying the light source device to optogenetics tool protein excitation spectrum measurement. According to the invention, the single-wave-pass optical filters are fixed on the optical filter wheel, and different optical filters are switched to the optical path of the full-spectrum light source through the rotation of the optical filter wheel, so that the system can output light with different wavelengths; the system can output a series of light with different wavelengths and the same photon flux by adjusting the power of a light source or installing a light reducing film on a light filter wheel; the output light is conducted to cells needing electrophysiological detection through the optical fiber, and when electrophysiological detection is carried out, the needed light can act on optogenetics tool protein expressed on a cell membrane. According to the light source device disclosed by the invention, the function of conveniently exciting the excitation spectrum of the optogenetics tool protein is realized by using the minimized equipment component, and the defect that the same function can be realized only by using complicated manual operation or expensive equipment with redundant functions in the traditional method is overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of optogenetics, and particularly to a light source device capable of outputting excitation light of different wavelengths and a method for detecting the excitation spectrum of optogenetic tool proteins using this light source device. Background Art

[0002] Optogenetics is a technology that uses genetic engineering methods to express photosensitive proteins of microorganisms in mammalian cells and changes the physiological state of cells using the photoactivity of photosensitive proteins. In the research of neuroscience, researchers often need to quickly, accurately, and reversibly modulate the excitatory state of neurons to study neural circuits and neural coding methods. Optogenetics perfectly meets this need and has become an important technology to promote the development of neurobiology. Not only in the field of neuroscience, but also important progress has been made in optogenetics in the fields of ophthalmology, metabolic regulation, cardiology, blood cell regulation, etc.

[0003] The progress of optogenetics is inseparable from the development of optogenetic tools. In 2003, Georg Nagel et al. first reported Channnelrhodopsin 2 (ChR2) and first confirmed that ChR2 can change the cell membrane potential. Two years later, Edward S Boyden et al. first expressed ChR2 on neurons cultured in vitro and successfully achieved optogenetic control of neurons, thus opening up optogenetics. Subsequently, light-gated anion channels GtACR1-2, light-gated sodium pumps KR2, light-gated potassium channels KCR1-2, etc. were reported and gradually applied to scientific research and clinical practice.

[0004] When developing and optimizing optogenetic tool proteins and using optogenetic tool proteins for optogenetic control and other applications, an important task is to detect the optimal excitation spectrum of optogenetic tool proteins. Only by understanding the optimal excitation spectrum of optogenetic tool proteins can appropriate light sources be used to maximize the effect of optogenetic tools.

[0005] However, due to the high requirements of optogenetic tools for the wavelength and intensity of light sources, existing light source devices are difficult to meet these requirements in terms of spectral coverage, light intensity stability, and wavelength switching. In addition, existing optogenetic tool protein excitation spectrum detection technologies usually rely on the combination of multiple independent devices, such as lasers, neutral density filter wheels, etc. They not only lack an integrated solution but also cannot be compatible with electrophysiological recording devices.

[0006] In summary, there is still a lack of integrated devices specifically for detecting the excitation spectrum of optogenetic tool proteins. Summary of the Invention

[0007] In view of the problem that there is a lack of integrated equipment in the prior art dedicated to detecting the excitation spectrum of optogenetic tool proteins, the present invention provides a light source device and a method for applying the same to measure the excitation spectrum of optogenetic tool proteins. The light source device of the present invention has the characteristics of small size and convenient operation, and can output a series of excitation lights with different wavelengths and the same number of photons respectively. The excitation light is then used to excite the optogenetic tool proteins on the cells, and these proteins will generate ion flow after being excited. As the excitation efficiency increases, the more ion flow is generated. The current generated by the ion flow can be detected by an electrophysiological device.

[0008] The technical solution adopted by the present invention is as follows:

[0009] I. A light source device for measuring the excitation spectrum of optogenetic tool proteins

[0010] The light source device includes a full-spectrum light source, a filter wheel, a wheel driver, a focusing lens, a three-axis fiber collimator, an input fiber, and an output fiber; the full-spectrum light source outputs raw light, the raw light is transmitted to the filter wheel through the input fiber, and is modulated by the filter wheel to generate excitation lights with different wavelengths and the same photon flux. The excitation light is converged into a light spot by the focusing lens, and the three-axis fiber collimator aligns the input end of the output fiber with the light spot. The converged excitation light is output from the output fiber and incident on the sample position; the filter wheel is in transmission connection with the wheel driver and is driven by the wheel driver.

[0011] A plurality of wheel holes are provided on the filter wheel, and the wheel holes are used to place single-bandpass filters and light attenuation components. A single-bandpass filter is installed in each wheel hole, and the single-bandpass filters in each wheel hole have different central wavelengths; the light attenuation component is used to attenuate the light beam generated by the single-bandpass filter arranged in the same wheel hole as itself, so that the excitation lights generated through different wheel holes have the same photon flux.

[0012] The light attenuation component is arranged on the incident light side of the corresponding single-bandpass filter; the single-bandpass filter is installed in the wheel hole; the light attenuation component is installed in the wheel hole.

[0013] The light attenuation component adopts a light attenuation filter film or a filter; all the single-bandpass filters adopt narrow-band filters.

[0014] In the filter wheel, the number of single-bandpass filters is 10, and the central wavelengths are respectively: 425nm ± 10nm, 450nm ± 10nm, 475nm ± 10nm, 500nm ± 10nm, 525nm ± 10nm, 550nm ± 10nm, 575nm ± 10nm, 600nm ± 10nm, 625nm ± 10nm, 650nm ± 10nm.

[0015] The light source device further includes an optical fiber adapter, a coaxial cage plate, and a lens sleeve; there are three coaxial cage plates in total, which are the first coaxial cage plate, the second coaxial cage plate, and the third coaxial cage plate in sequence along the optical path; the input optical fiber is connected to one end of the optical fiber adapter, and the other end of the optical fiber adapter is centered and directly opposite to the light input port of the filter wheel through the first coaxial cage plate. The light output port of the filter wheel is centered and directly opposite to the second coaxial cage plate and the focusing lens. The focusing lens is fixed in the second coaxial cage plate through the lens sleeve; a three-axis optical fiber collimator is installed in the third coaxial cage plate. The three-axis optical fiber collimator is connected to the output optical fiber, and the three-axis optical fiber collimator is used to align the input end of the output optical fiber with the focal point of the focusing lens.

[0016] The full-spectrum light source includes a light source and a light source brightness regulator; the light source brightness regulator is used to adjust the input voltage or input current of the light source; the output spectrum of the light source is 350nm - 750nm, and the light source is a tungsten lamp, a xenon lamp, or an LED lamp.

[0017] The sample is placed on the sample stage of the electrophysiological device. The output optical fiber is detachably installed on the electrophysiological device. The output end of the output optical fiber is located above the sample. After the excitation light is output from the output optical fiber and incident on the sample position, a light spot is formed at the sample position. The area of the light spot is larger than the area of the sample and covers the sample.

[0018] II. A method for measuring the excitation spectrum of a optogenetic tool protein using the above light source device. The method includes the following steps:

[0019] S1) Obtain single-bandpass filters with corresponding central wavelengths for each excitation wavelength and install them in the respective rotating holes of the filter wheel.

[0020] S2) At the same output power of the full-spectrum light source, use the rotating wheel driver to sequentially switch each single-bandpass filter to the light passing optical path of the filter wheel. Use a optical power meter to collect the light intensity of the excitation light corresponding to each single-bandpass filter respectively, and obtain the photon flux according to the wavelength and light intensity of each beam of excitation light.

[0021] Use the rotating wheel driver to switch the single-bandpass filter corresponding to the excitation light with the minimum photon flux to the light passing optical path of the filter wheel. Turn on the full-spectrum light source, and use the light source brightness regulator to adjust the output power of the full-spectrum light source to adjust the light intensity of the excitation light to 50 - 200 μW / mm 2 , and obtain the photon flux at this time as the target photon flux.

[0022] Determine the target light intensity corresponding to each of the remaining single-bandpass filters according to the target photon flux.

[0023] S3) using a wheel driver to switch the remaining single-wave pass filters to the light path of the filter wheel, and using an optical power meter to obtain the light intensity of the excitation light corresponding to each single-wave pass filter;

[0024] According to the illumination intensity of the excitation light and the target light intensity corresponding to each single-wave pass filter, the corresponding attenuation coefficient is determined, and the dimming component is selected according to the attenuation coefficient and installed in the corresponding rotating wheel hole;

[0025] S4) placing the sample on the sample stage of the electrophysiological device, moving and fixing the output end of the output optical fiber above the sample, so that the excitation light is output from the output optical fiber and incident on the sample position, and adjusting the distance between the output end of the output optical fiber and the sample so that the light spot area is larger than the sample area and covers the sample;

[0026] S5) electrically connecting any cell in the sample to the current detection device, using a wheel driver to sequentially switch each single-wave pass filter to the light path of the filter wheel, and when all single-wave pass filters have completed switching and electrical signal collection has been completed, the measurement of the cell is completed;

[0027] S6) completing the measurement of at least six cells according to step S5;

[0028] S7) For the measurement result of each cell, the difference between the current collected by the cell under each single-wavelength pass filter and the current in the light-free state is obtained as the photocurrent at the corresponding wavelength. After the photocurrent of the cell at each wavelength is normalized, a curve graph of the photocurrent changing with wavelength is obtained as the excitation spectrum graph.

[0029] The sample mainly consists of a plurality of cells containing the optogenetic tool protein to be detected.

[0030] The beneficial effects of the present invention are as follows:

[0031] The present invention proposes a device that can switch light sources of different wavelengths. The device can also keep the photon flux of the output excitation light consistent by adjusting the brightness of the light source and the use of a light-reducing film. The device can be used in conjunction with an electrophysiological device to detect the excitation spectrum of an optogenetic tool protein. The device of the present invention has the necessary components for measuring the excitation spectrum of an optogenetic tool protein and is easy to operate. Compared with the traditional method of frequently using plug-in and unplugging to switch filters in a slot, the present invention only needs to control the rotation of a wheel to achieve the switching of output light of different wavelengths, which not only brings convenience in operation, but also reduces experimental errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The present invention relates to a diagram of key components;

[0033] Figure 2The excitation spectrum diagram of HcKCR1 detected using the present invention.

[0034] In the figure: 1. Full-spectrum light source, 101. Light source, 102. Light source brightness regulator; 2. Input optical fiber; 3. Fiber optic adapter; 4. Coaxial cage plate; 5. Filter wheel, 501. Single-bandpass filter, 502. Light attenuation component; 6. Wheel driver; 7. Lens sleeve; 8. Focusing lens; 9. Three-axis fiber optic collimator; 10. Output optical fiber. Detailed implementation manners

[0035] In order to deeply reveal the core purpose, technical solution and its remarkable advantages of the present invention, hereby in combination with the attached drawings, the technical solutions in the embodiments of the present invention are elaborated in detail. It should be clear that the embodiments presented here are only a part of numerous examples of the present invention, rather than all of them. The components shown and described in the drawings can be arranged and designed according to a variety of different configurations. Importantly, the following detailed description of the embodiments of the present invention is not intended to limit its protection scope. On the contrary, it is intended to be an example of certain selected embodiments of the present invention. All other implementation manners proposed by those skilled in the art based on the present invention without creative efforts shall be regarded as falling within the protection scope of the present invention.

[0036] The following further explains and interprets the relevant terms in the present invention:

[0037] "Optogenetics" in the present invention refers to: a discipline that transfers light-sensitive proteins into specific host cells through genetic engineering methods, and uses light to change the state of light-sensitive proteins, thereby affecting the physiological state of host cells.

[0038] "Optogenetic tool protein" in the present invention refers to: light-sensitive proteins used in the field of optogenetics, including but not limited to light-gated ion channels, light-gated ion pumps, light-gated enzymes, etc.

[0039] "Excitation spectrum" in the present invention refers to: when using an electrophysiological device to detect the photocurrent of a light-sensitive protein, light of different wavelengths will cause differences in the opening of the light-sensitive protein, and thus show different magnitudes of current caused by ion flow. After the current signal is amplified and collected by the electrophysiological device, a tiny current caused by light can be recorded. The curve plotted by the excitation light of different wavelengths and the photocurrent of the light-sensitive protein is the excitation spectrum of the light-sensitive protein.

[0040] The first aspect of the present invention provides a light source device for measuring the excitation spectrum of an optogenetic tool protein.

[0041] The light source device includes a full-spectrum light source 1, an input optical fiber 2, a filter wheel 5, a wheel driver 6, a focusing lens 8, a three-axis fiber collimator 9, and an output optical fiber 10. The focusing lens 8 is used to focus the scattered light to a point. The filter wheel 5 is used to modulate the original light into excitation light with different wavelengths but the same photon flux.

[0042] Specifically, the beam transmission path inside the light source device of the present invention is as follows: The full-spectrum light source 1 outputs the original light, which is transmitted through the input optical fiber 2 to the filter wheel 5. The filter wheel 5 modulates the original light to generate excitation light with different wavelengths but the same photon flux. The excitation light is converged into a light spot by the focusing lens 8. The three-axis fiber collimator 9 aligns the input end of the output optical fiber 10 with the light spot. The converged excitation light is output from the output optical fiber 10 and incident on the sample position. The filter wheel 5 is in driving connection with the wheel driver 6 and is driven by the wheel driver 6 to rotate around its own axis.

[0043] Among them, "the three-axis fiber collimator 9 aligns the input end of the output optical fiber 10 with the light spot" means that the position of the input end of the output optical fiber 10 is adjusted by the three-axis fiber collimator 9 so that the focal point of the focusing lens 8 is aligned with the input end of the output optical fiber 10, which can ensure that the light energy of the light spot can be coupled into the optical fiber to the maximum extent.

[0044] Among them, the excitation light of each wavelength has the same photon flux, that is, the light source device of the present invention can output excitation light with different wavelengths but the same light flux. At the same power, different wavelengths have different photon energies, resulting in inconsistent numbers of photons per unit time. In order to ensure that the number of photons of each wavelength is the same, the present invention makes the photon fluxes of the excitation lights of each wavelength consistent through this light source device.

[0045] In the light source device of the present invention, the implementation method for the filter wheel 5 to modulate the original light into excitation light with different wavelengths but the same photon flux is as follows: A plurality of wheel holes are provided on the filter wheel 5, and the wheel holes are arranged at equal intervals along the circumferential direction of the filter wheel 5. The axis of the filter wheel 5, the light passing optical path inside the filter wheel 5, and the axis of the wheel holes are parallel to each other. The light passing optical path inside the filter wheel 5, the light input port, and the light output port are coaxially arranged.

[0046] The filter wheel 5 includes a plurality of single-wavelength-pass filters 501 and a plurality of light attenuation components 502. The wheel holes are used to place the single-wavelength-pass filters 501 and the light attenuation components 502. Specifically: One single-wavelength-pass filter 501 is installed in each wheel hole, and the single-wavelength-pass filters 501 in each wheel hole have different central wavelengths; so that after the original light passes through different wheel holes, light beams with different wavelengths are formed. The light attenuation component 502 is used to attenuate the light beam generated by the single-wavelength-pass filter 501 arranged in the same wheel hole as itself, so that the excitation light generated through different wheel holes has the same photon flux.

[0047] In specific implementation, the number of light attenuation components 502 in each runner hole can be zero. For example, in an alternative embodiment, if the number of single-bandpass filters 501 is N, the number of light attenuation components 502 is N - 1. Except for the single-bandpass filter 501 corresponding to the light with the highest required light intensity after filtering, one light attenuation component 502 is correspondingly provided for each of the other single-bandpass filters 501.

[0048] Preferably, for the runner holes provided with single-bandpass filters 501 and corresponding light attenuation components 502, the light attenuation components 502 are arranged on the incident light side of the corresponding single-bandpass filters 501; the single-bandpass filters 501 are detachably installed in the runner holes; the light attenuation components 502 are detachably installed in the runner holes.

[0049] Based on the above preferred solution, the process of using the filter wheel 5 to modulate the original light into an excitation light with a target excitation wavelength and a target photon flux is as follows: First, select a single-bandpass filter 501 with a center wavelength the same as the target excitation wavelength from the single-bandpass filters 501 installed on the filter wheel 5 and use it as the target single-bandpass filter 501. Subsequently, rotate the filter wheel 5 to align the runner hole with the target single-bandpass filter 501 with the light passing optical path, that is, switch the target single-bandpass filter 501 to the light passing optical path. At this time, if no light attenuation component 502 is provided in the runner hole, the output light of the full-spectrum light source 1 passes through the input optical fiber 2 and the target single-bandpass filter 501 in sequence to generate excitation light, and the excitation light is output from the output optical fiber 10 and incident on the sample position; if a light attenuation component 502 is provided in the runner hole, the output light of the full-spectrum light source 1 passes through the input optical fiber 2, the light attenuation component 502, and the target single-bandpass filter 501 in sequence to generate excitation light, and the excitation light is output from the output optical fiber 10 and incident on the sample position.

[0050] The light attenuation component 502 refers to a thin film or filter that can weaken the light intensity. The light attenuation component 502 uses a light attenuation filter film or filter. The light attenuation filter film is usually a black or gray plastic sheet, and the filter is made of glass. Both can absorb a certain ratio of the passing light. In specific implementation, each light attenuation component 502 can select a light attenuation filter film with the same transmittance, and the overall transmittance can be precisely controlled by adjusting the number of layers of the light attenuation filter film included in the light attenuation component 502. For example, assuming that the transmittance of each light attenuation filter film is T, when n layers of light attenuation filter films are stacked to form a light attenuation component 502, the transmittance T of the light attenuation component 502 total can be expressed as T n . By increasing or decreasing the number of layers of the light attenuation filter film included in each light attenuation component 502, the transmittance of the light attenuation component 502 can be flexibly adjusted so that the excitation lights with different wavelengths generated through each runner hole have the same photon flux.

[0051] Preferably, the single-wavelength pass filter 501 all adopts a narrow-band filter. A narrow-band filter refers to a filter that only allows light of a specific wavelength to pass through.

[0052] As an alternative embodiment of the present invention, in the filter wheel 5, the number of single-wavelength pass filters 501 is 10, and the central wavelengths are respectively: 425 nm ± 10 nm, 450 nm ± 10 nm, 475 nm ± 10 nm, 500 nm ± 10 nm, 525 nm ± 10 nm, 550 nm ± 10 nm, 575 nm ± 10 nm, 600 nm ± 10 nm, 625 nm ± 10 nm, 650 nm ± 10 nm. The number of light attenuation components 502 is 9. Except for the single-wavelength pass filter 501 corresponding to the light with the highest required light intensity after filtering, light attenuation components 502 are correspondingly arranged for other single-wavelength pass filters 501.

[0053] Optionally, the way the filter wheel 5 is drivingly connected to the wheel driver 6 is as follows: the filter wheel 5 is annular, with a gear at the center, and the gear can be drivingly connected to the transmission shaft of the wheel driver 6. The wheel driver 6 can adopt a motor.

[0054] Furthermore, the wheel driver 6 can be electrically connected to an accessory driver program and the number of rotation turns can be precisely controlled by the accessory driver program.

[0055] Specifically, the sample is placed on the sample stage of the electrophysiological device, the output optical fiber 10 is detachably installed on the electrophysiological device, the output end of the output optical fiber 10 is located above the sample. After the excitation light is output from the output optical fiber 10 and incident on the sample position, a light spot is formed at the sample position.

[0056] Preferably, at the sample position, the area of the light spot preferably covers the area of the sample and covers the sample.

[0057] Furthermore, the light source device further includes an optical fiber adapter 3, a first coaxial cage plate, a second coaxial cage plate, a third coaxial cage plate, and a three-axis optical fiber collimator 9. As Figure 1 shown, the three coaxial cage plates are, in the optical path (from left to right), the first coaxial cage plate, the second coaxial cage plate, and the third coaxial cage plate in sequence. The input optical fiber 2 is connected to one end of the optical fiber adapter 3, the other end of the optical fiber adapter 3 is connected to the light input port of the filter wheel 5 through the first coaxial cage plate, and the light output port of the filter wheel 5 is centered and opposite to the second coaxial cage plate 4 and the focusing lens 8. The third coaxial cage plate is used to fix the three-axis optical fiber collimator 9. The three-axis optical fiber collimator 9 is connected to the output optical fiber 10.

[0058] In the specific embodiment of the present invention, all three coaxial cage plates adopt 30 mm coaxial cage plates.

[0059] Optionally, the input optical fiber 2 and the fiber optic adapter 3 are connected by an FC or SMA bayonet, and the three-axis fiber optic collimator 9 and the output optical fiber 10 are connected by an FC or SMA bayonet.

[0060] Furthermore, the full-spectrum light source 1 should have a voltage regulator for adjusting the light source voltage or a current regulator for adjusting the light source current, as well as other means for adjusting the light source brightness.

[0061] Preferably, the full-spectrum light source 1 includes a light source 101 and a light source brightness regulator 102. The input end of the light source brightness regulator 102 is electrically connected to an external power supply, and the output end is electrically connected to the light source 101, for adjusting the input voltage or input current of the light source 101.

[0062] Preferably, the output spectrum of the light source 101 is 350 nm to 750 nm.

[0063] Preferably, the light source 101 is a tungsten lamp, a xenon lamp or an LED lamp.

[0064] Preferably, the input optical fiber 2 and the output optical fiber 10 can be made of light-guiding optical fibers such as PMMA optical fibers or quartz optical fibers.

[0065] Furthermore, the light source device further includes a bracket and a chassis. The bracket is used to fix the coaxial cage plate 4 and the runner driver 6 on the chassis. The full-spectrum light source 1 is fixed on the chassis by screws. The chassis is an optical breadboard for convenient fixing, etc.

[0066] The second aspect of the present invention provides a method for measuring the excitation spectrum of an optogenetic tool protein.

[0067] It includes the following steps:

[0068] S1) In the excitation wavelength range, select a plurality of different excitation wavelengths, and obtain a single-bandpass filter 501 with a corresponding central wavelength according to each excitation wavelength, and install the obtained plurality of single-bandpass filters 501 in the respective runner holes of the filter wheel 5;

[0069] S2) At the same output power of the full-spectrum light source 1, use the runner driver 6 to sequentially switch each single-bandpass filter 501 to the light-passing optical path of the filter wheel 5, use a optical power meter to collect the light intensity of the excitation light corresponding to each single-bandpass filter 501, and obtain the photon flux according to the wavelength and light intensity of each beam of excitation light;

[0070] Use the turret driver 6 to switch the single-bandpass filter 501 corresponding to the excitation light with the minimum photon flux to the light path of the turret 5, turn on the full-spectrum light source 1, and use the light source brightness regulator 102 to adjust the output power of the full-spectrum light source 1 to adjust the illumination intensity of the excitation light to 50-200 μW / mm 2 , and obtain the photon flux at this time as the target photon flux;

[0071] Determine the target light intensity corresponding to each of the remaining single-bandpass filters 501 according to the target photon flux;

[0072] S3) Use the turret driver 6 to switch each of the remaining single-bandpass filters 501 to the light path of the turret 5 respectively, and use a optical power meter to obtain the illumination intensity of the excitation light corresponding to each single-bandpass filter 501;

[0073] According to the illumination intensity of the excitation light corresponding to each single-bandpass filter 501 and the target light intensity, determine the corresponding attenuation coefficient, select a light attenuation component 502 according to the attenuation coefficient, and install the selected light attenuation component 502 into the corresponding turret hole to attenuate the illumination intensity of the excitation light corresponding to the single-bandpass filter 501 to the target light intensity;

[0074] S4) Place the sample on the sample stage of the electrophysiological device, move and fix the output end of the output optical fiber 10 above the sample, so that the excitation light is output from the output optical fiber 10 and incident on the sample position, and adjust the distance between the output end of the output optical fiber 10 and the sample so that the spot area is larger than the area of the sample and covers the sample;

[0075] Among them, the sample mainly consists of multiple cells containing the optogenetic tool protein to be detected;

[0076] S5) Electrically connect any one of the cells in the sample to the current detection device to collect the electrical signals generated by the cell during the measurement process. Use the turret driver 5 to sequentially switch each single-bandpass filter 501 to the light path of the turret 5. Each single-bandpass filter 501 stays for 1 s and then switches to the next single-bandpass filter 501. When all single-bandpass filters 501 have been switched and the electrical signal collection is completed, the measurement of the cell is completed;

[0077] S6) Measure the next cell according to step S5 until at least six cells have been measured, and the measurement ends;

[0078] S7) For the measurement results of each cell, obtain the difference between the current collected by the cell under each single-bandpass filter 501 and the current in the light-off state as the photocurrent at the corresponding wavelength. After normalizing the photocurrents of the cell at each wavelength, obtain a curve graph of the photocurrent changing with the wavelength as the excitation spectrum graph.

[0079] Specific embodiments of the present invention are as follows:

[0080] Embodiment 1

[0081] The following describes an embodiment of using the present device to measure the excitation spectrum of the optogenetic tool protein HcKCR1 in conjunction with the accompanying drawings. As Figure 1 shown, the device of the present invention is used in combination with an electrophysiological device to measure the excitation spectrum of HcKCR1. The electrophysiological device used in this embodiment is an amplifier (npi, TEC-03x) and a digital-to-analog converter (national instrument, USB-6221).

[0082] This embodiment includes the following steps:

[0083] 1. Preliminary preparation

[0084] 1.1 Preparation of the light source device:

[0085] S1) Fix 10 narrowband filters in the holes reserved on the filter wheel respectively. The transmission wavelengths of the filters are 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, and 650 nm respectively, and the central wavelength width is ±10 nm.

[0086] S2) Determine the target photon flux and the target light intensity corresponding to each single-bandpass filter 501: In this embodiment, taking the light intensity of the output light at 425 nm as 150 μW / mm 2 as an example, according to the following formula, it can be known that the light intensity is inversely proportional to the light wavelength:

[0087]

[0088] In the formula, I is the light intensity, Φ is the photon flux, λ is the light wavelength, h is Planck's constant, and c is the speed of light.

[0089] Since Planck's constant, the speed of light, and the spot area are fixed values, the light intensity corresponding to each wavelength should be as shown in Table 1:

[0090] Table 1

[0091]

[0092] Use a turntable driver to switch each of the remaining single-bandpass filters to the light path of the filter turntable, and use a optical power meter to obtain the illumination intensity of the excitation light corresponding to each single-bandpass filter; determine the corresponding attenuation coefficient according to the illumination intensity of the excitation light corresponding to each single-bandpass filter and the target light intensity, select a light attenuation component according to the attenuation coefficient, and install the light attenuation component into the corresponding turntable hole to attenuate the illumination intensity of the excitation light corresponding to the single-bandpass filter to the target light intensity;

[0093] S3) Use the turntable to switch the turntable hole configured with the 450nm filter to the light path, install one or more layers of light attenuation filters on the turntable hole, and adjust the light intensity to 141.7 μW / mm 2 。

[0094] Adjust the other turntable holes in the same way so that the light intensities of different wavelengths output are as shown in Table 1 above.

[0095] 1.2. Preparation of experimental materials:

[0096] Inject 20 ng of mRNA encoding HcKCR1 into Xenopus laevis oocytes by microinjection. Subsequently, add all-trans retinoic acid with a final concentration of 1 μM to the culture medium, place it in the dark in an 18°C incubator, and perform electrophysiological detection after 48 hours.

[0097] 1.3. Preparation of electrophysiological equipment:

[0098] S4) Before starting the detection, preheat the equipment for half an hour. Insert the output optical fiber of the present invention into the corresponding position of the recording chamber so that the optical fiber spot covers the position where the oocytes are placed. Set the recording time to 2 min.

[0099] S5) Electrophysiological detection of photocurrent process:

[0100] S5.1) The bath solution used for detection is Ori solution (110 mM NaCl, 5 mM KCl, 5 mM HEPES, 1 mM MgCl2, 2 mM CaCl2, pH = 7.6), and this solution is continuously perfused during the detection process. Place the oocytes.

[0101] S5.2) Use the manipulator to insert the current electrode and voltage electrode into the bath solution respectively, and zero the two electrode potentials respectively. Turn on the full-spectrum light source for preheating, and turn the turntable to the non-light-passing state.

[0102] S5.3) Insert the current electrode and voltage electrode into the oocytes respectively, clamp the cell membrane potential of the oocytes at -60 mV. Start recording, and after 5 s, switch the 425nm filter hole of the turntable to the light path and stay for one second.

[0103] S5.4) Rotate the turntable. During the rotation, the output optical fiber is in a non-light-passing state, and at this time, the photocurrent is 0 A. Wait for 1 s after the 450 nm filter hole is switched to the optical path, and the photocurrent is generated again.

[0104] S5.5) Continue to rotate the turntable, and sequentially switch the filter holes to 650 nm to end the measurement.

[0105] S6) When measuring the next oocyte, use the reverse order method, that is, sequentially measure from the 650 nm filter hole to 425 nm. At least measure n = 6 groups of data to end the test.

[0106] 1.4. Data processing:

[0107] S7) The absolute value of the current in the light-passing state minus the current in the non-light state is the photocurrent. Each photocurrent value is normalized by dividing it by the largest photocurrent in the group of data. Using the normalized values as the ordinate and the corresponding wavelengths as the abscissa, a curve is plotted. This curve graph is the excitation spectrum of HcKCR1. The results are as Figure 2 shown. It can be seen from the figure that the optimal excitation light of HcKCR1 is in the green light region, which is consistent with the excitation spectrum of HcKCR1 in the published papers.

[0108] The above specific embodiments are used to explain and illustrate the present invention, rather than to limit the present invention. Any modifications and changes made within the spirit and scope of the claims of the present invention fall within the protection scope of the present invention.

[0109] The above are only the preferred embodiments of the present invention. Therefore, all equivalent changes or modifications made according to the structures, features, and principles described in the scope of the present invention patent application are included in the scope of the present invention patent application.

Claims

1. A light source device for measuring the excitation spectrum of optogenetic tool proteins, characterized in that: The light source device comprises a full-spectrum light source (1), a filter wheel (5), a wheel driver (6), a focusing lens (8), a three-axis fiber collimator (9), an input fiber (2), and an output fiber (10); the full-spectrum light source (1) outputs original light, which is transmitted to the filter wheel (5) via the input fiber (2), modulated by the filter wheel (5) to generate excitation light with different wavelengths and the same photon flux, the excitation light is converged into a light spot via the focusing lens (8), the three-axis fiber collimator (9) aligns the input end of the output fiber (10) with the light spot, and the converged excitation light is output from the output fiber (10) and incident on a sample position; the filter wheel (5) is transmission-connected to the wheel driver (6) and driven by the wheel driver (6).

2. The light source device according to claim 1, characterized in that: The filter wheel (5) is provided with a plurality of wheel holes, the wheel holes being used to place single-wave pass filters (501) and dimming components (502), each wheel hole being provided with a single-wave pass filter (501), and the single-wave pass filters (501) in each wheel hole having a different central wavelength; the dimming component (502) is used to attenuate a light beam generated by a single-wave pass filter (501) arranged in the same wheel hole as the single-wave pass filter (501), so that excitation light generated via different wheel holes has the same photon flux.

3. The light source device according to claim 2, characterized in that: The light reduction component (502) is arranged on the light incident side of the corresponding single-wave pass filter (501); the single-wave pass filter (501) is embedded in the rotating wheel hole; and the light reduction component (502) is embedded in the rotating wheel hole.

4. The light source device according to claim 2, characterized in that: The light reduction component (502) adopts a light reduction filter film or a filter; the single wave pass filter (501) adopts a narrow band filter.

5. The light source device according to claim 2, characterized in that: In the filter wheel (5), the number of single-wave pass filters (501) is 10, and the central wavelengths are respectively: 425nm±10nm, 450nm±10nm, 475nm±10nm, 500nm±10nm, 525nm±10nm, 550nm±10nm, 575nm±10nm, 600nm±10nm, 625nm±10nm, and 650nm±10nm.

6. The light source device according to claim 1, characterized in that: The light source device further comprises an optical fiber adapter (3), a coaxial cage plate (4) and a lens sleeve (7); there are three coaxial cage plates (4), which are the first coaxial cage plate, the second coaxial cage plate and the third coaxial cage plate in sequence along the optical path; the input optical fiber (2) is connected to one end of the optical fiber adapter (3), the other end of the optical fiber adapter (3) is centered on the light inlet of the filter wheel (5) through the first coaxial cage plate, the light outlet of the filter wheel (5) is centered on the second coaxial cage plate and the focusing lens (8), and the focusing lens (8) is fixed in the second coaxial cage plate through the lens sleeve (7); A three-axis fiber collimator (9) is installed in the third coaxial cage plate. The three-axis fiber collimator (9) is connected to the output optical fiber (10). The three-axis fiber collimator (9) is used to align the input end of the output optical fiber (10) with the focus of the focusing lens (8).

7. The light source device according to claim 1, characterized in that: The full-spectrum light source (1) comprises a light source (101) and a light source brightness adjuster (102); the light source brightness adjuster (102) is used to adjust an input voltage or an input current of the light source (101); the output spectrum of the light source (101) is 350 nm to 750 nm, and the light source (101) is a tungsten lamp, a xenon lamp or an LED lamp.

8. The light source device according to claim 1, characterized in that: The sample is placed on a sample stage of an electrophysiological device, the output optical fiber (10) is detachably mounted on the electrophysiological device, the output end of the output optical fiber (10) is located above the sample, and after the excitation light is output from the output optical fiber (10) and incident on the sample position, a light spot is formed at the sample position, and the area of ​​the light spot is larger than the area of ​​the sample and covers the sample.

9. A method for measuring the excitation spectrum of an optogenetic tool protein using the light source device according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1) obtaining a single-wave pass filter (501) of a corresponding central wavelength according to each excitation wavelength, and installing the single-wave pass filter in each wheel hole of the filter wheel (5); S2) under the same output power of the full-spectrum light source (1), using a wheel driver (6) to sequentially switch each single-wave pass filter (501) to the light path of the filter wheel (5), using an optical power meter to respectively collect the light intensity of the excitation light corresponding to each single-wave pass filter (501), and obtaining the photon flux according to the wavelength and light intensity of each beam of excitation light; The single-wave filter (501) corresponding to the excitation light with the minimum photon flux is switched to the light path of the filter wheel (5) using the wheel driver (6), the full-spectrum light source (1) is turned on, and the output power of the full-spectrum light source (1) is adjusted using the light source brightness adjuster (102), so that the illumination intensity of the excitation light is adjusted to 50 to 200 μW / mm 2 , obtain the photon flux at this time and use it as the target photon flux; Determining the target light intensity corresponding to each of the remaining single-wave pass filters (501) according to the target photon flux; S3) using a wheel driver (6) to switch the remaining single-wave pass filters (501) to the light path of the filter wheel (5), and using an optical power meter to obtain the light intensity of the excitation light corresponding to each single-wave pass filter (501); Determine the corresponding attenuation coefficient according to the illumination intensity of the excitation light corresponding to each single-wave pass filter (501) and the target light intensity, select the dimming component (502) according to the attenuation coefficient and install it in the corresponding rotating wheel hole; S4) placing the sample on a sample stage of an electrophysiological device, moving and fixing the output end of the output optical fiber (10) above the sample, so that the excitation light is output from the output optical fiber (10) and incident on the sample position, and adjusting the distance between the output end of the output optical fiber (10) and the sample so that the light spot area is larger than the sample area and covers the sample; S5) electrically connecting any cell in the sample to the current detection device, using the wheel driver (5) to sequentially switch each single-wave pass filter (501) to the light path of the filter wheel (5), and when all single-wave pass filters (501) have completed switching and electrical signal collection has been completed, the measurement of the cell is completed; S6) completing the measurement of at least six cells according to step S5; S7) For the measurement result of each cell, the difference between the current collected by the cell under each single-wavelength filter (501) and the current in the light-free state is obtained as the photocurrent at the corresponding wavelength. After the photocurrent of the cell at each wavelength is normalized, a curve graph of the photocurrent changing with wavelength is obtained as the excitation spectrum graph.

10. The method for measuring the excitation spectrum of optogenetic tool proteins according to claim 9, characterized in that: The sample mainly consists of a plurality of cells containing the optogenetic tool protein to be detected.

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