A multi-wavelength arrayed light modulation and detection device for optogenetics

Abstract

The application discloses a kind of multi-wavelength array type light regulation and detection device for optical genetics, belong to the field of bioengineering and automatic control, comprising: array light source, mechanical control platform, imaging device and control module;Mechanical control platform is provided with microwell plate, and the microwell plate includes a plurality of array culture wells for cell culture;Control module is connected with array light source;Array light source outputs a plurality of groups of sub-beams distributed in array, and the position of the plurality of groups of sub-beams corresponds to the culture well of different position on microwell plate respectively;The light of each group of sub-beams has different target parameters, including wavelength, illumination intensity and time.The multi-wavelength array type light regulation and detection device for optical genetics disclosed in the application can be used to construct multi-wavelength array type light regulation experimental platform with controllable illumination intensity and time, and further tap the potential of optical genetics in cell high-throughput metabolic regulation.

Description

Technical Field

[0001] This application relates to the fields of bioengineering and automatic control, and more specifically, to a multi-wavelength array-type light modulation and detection device for optogenetics. Background Technology

[0002] Precise regulation of gene expression is crucial for studying various life phenomena in organisms. Current methods of metabolic regulation have limitations; conventional methods primarily utilize chemical inducers. While these inducers can efficiently induce the expression of exogenous genes, they also have drawbacks such as toxicity and the inability to spatially and specifically regulate gene expression. Furthermore, chemical inducers suffer from high costs, inability to be switched off, cytotoxicity, and the inability to achieve dynamic regulation, and their modification is time-consuming and labor-intensive. Optogenetics, on the other hand, is a new technology that combines light control with genetics for cell biology research. It involves introducing genetically encoded optogenetic proteins into cells, using light to directly, specifically, rapidly, remotely, reversibly, quantitatively, and locally in real-time control of intracellular molecular activities.

[0003] Compared to chemically induced regulation, optogenetic regulation has advantages primarily in its low toxicity, ease of acquisition, ease of manipulation, and high spatiotemporal resolution. Optogenetic proteins are a class of proteins found in nature that can regulate their function and activity in response to external light signals. From the perspective of chromophores and photochemistry, currently discovered optogenetic proteins are classified into six categories: LOV receptors, xanthopsins, phytochromes, blue light sensors using flavin adenine dinucleotide (BLUF), cryptochromes, and rhodopsins. The first five are water-soluble proteins, while the sixth is a membrane protein. The regulatory wavelengths of these six naturally occurring optogenetic proteins are distributed differentially within the ultraviolet-visible light range (300-700 nm), therefore, research on them involves light regulation at different wavelengths.

[0004] Meanwhile, light-controlled gene expression mechanisms can be categorized into five main types: interactions between photoreceptor proteins and their ligands; photoinduced signaling cascade pathways; two-component systems; allosteric transformation of the LOV domain; and photoinduced homodimerization. Among these, unit-based photoisomerization proteins gain or lose their original steric repression through conformational changes, such as LOV, Dronpa, and UVR8. Specifically, when a chromophore absorbs a photon, its structure changes, leading to conformational changes in the closely linked protein. At this point, the optogenetic protein is activated, regulating the expression of downstream target genes. In addition, it also involves the sequential or simultaneous irradiation of light of different wavelengths. For example, the mechanism of action of photosensitive binding proteins based on dual elements is that after absorbing light energy, the conformation of the optogenetic protein changes, enabling it to bind to optogenetic protein ligands, effectors or other proteins, such as the dual-element systems of CRY-CIB1 (450-480nm), PhyB-PIF (490nm / 390nm) and Dronpa (650nm / 750nm). The activation and reversal of the latter two require two different wavelengths of light.

[0005] Currently, optogenetic protein research typically requires light regulation using different wavelengths, light intensities, durations, irradiation sequences, and parameter combinations to provide external variables. Furthermore, studies involving optogenetic protein gene modification, optogenetic protein element screening, and microbial metabolic pathway optimization often involve combinations of multiple biological internal variables. Therefore, many different parameter experiments often need to be designed, and considering parallel comparisons and parameter gradients, the space of parameter variables to be tried and searched is extremely large. Traditional darkroom illumination can usually only provide a limited number of light regulation conditions at a time, such as a single wavelength, a limited number of light intensities, or light durations; this cannot meet the needs of the increasingly sophisticated optogenetic research. Therefore, there is an urgent need for devices and methods that provide parallel light regulation techniques such as adjustable light intensity (0.1-10 mW / cm²), controllable illumination time (from seconds to hours), and selectable multi-wavelength light within a single experimental group, while also considering compatibility with the sizes of commonly used experimental consumables such as petri dishes and microplates. Summary of the Invention

[0006] This application provides a multi-wavelength array-type light modulation and detection device for optogenetics, aiming to construct a multi-wavelength array-type light modulation experimental platform with controllable light intensity and time, thereby exploring the potential of optogenetics in high-throughput metabolic regulation of cells.

[0007] This application provides a multi-wavelength array-type light modulation and detection device for optogenetics, including:

[0008] Array-type light source, mechanical control platform, imaging device and control module;

[0009] The array-type light source is located on one side of the mechanical control platform, and the imaging device is located on the side of the mechanical control platform away from the light source. The array-type light source, the mechanical control platform, and the imaging device are on the same straight line.

[0010] The mechanical control platform is equipped with a microplate, which includes multiple culture wells for cell culture.

[0011] The control module is connected to the array light source, and the control module includes a control circuit board, which includes a light source driver.

[0012] The light source driver is used to enable the array light source to output multiple sets of sub-beams distributed in an array, and the positions of the multiple sets of sub-beams correspond to the culture wells at different positions on the microplate.

[0013] Each group of sub-beams has different target parameters, and the sub-beams are emitted from the array of light sources toward the microporous plate;

[0014] The imaging device is used to perform in-situ detection of cells cultured on the microplate.

[0015] Optionally, the target parameters include light wavelength, light intensity, pulse duty cycle, and illumination time.

[0016] Optionally, the array light source includes multiple sets of sub-light sources, and multiple light source drivers are provided, with each set of light source drivers connected to multiple sets of sub-light sources respectively;

[0017] The control circuit board also includes a microcontroller, which is connected to a plurality of the light source drivers and is used to send light modulation commands to the light source drivers.

[0018] The light source driver responds to the light modulation command by selecting and turning on the sub-light source of the target wavelength, driving the sub-light source to generate the corresponding sub-beam, and adjusting the light intensity, pulse duty cycle and illumination time of the sub-light source according to the light modulation command.

[0019] Optionally, the sub-light source includes multiple micro light-emitting diodes, and the wavelengths of the light emitted by the array of multiple micro light-emitting diodes in each group of the sub-light sources are different.

[0020] Optionally, the array-type light source includes a source light source, an optical template, and a lens group;

[0021] The optical template, the mechanical control platform, and the imaging device are on the same straight line, the source light source is located to the side of the optical template, and the lens group is disposed between the source light source and the optical template.

[0022] The control module is connected to the optical template, and the control module is used to send adjustment commands to the optical template;

[0023] The optical template is used to adjust the target parameters of multiple sub-beams emitted by the source light source according to the adjustment command, and to project the adjusted sub-beams toward the mechanical control platform.

[0024] The light source driver is connected to the source light source and is used to drive the source light source to emit the multiple sets of sub-beams and to adjust the wavelength of the light emitted by the sub-beams.

[0025] Optionally, the optical template includes a digital reflector, which is disposed on the side of the mechanical control platform away from the imaging device, and the digital reflector is tilted.

[0026] The light source includes multiple light-emitting diodes with different wavelengths and a condenser lens, and the condenser lens focuses the light from the light-emitting diodes onto the lens group;

[0027] The digital reflector is connected to the control module. The digital reflector is used to control the digital reflector to addressably adjust the reflection of light from the source light source according to the adjustment command of the control module, and to project multiple sets of sub-beams distributed in an array.

[0028] The digital reflector is used to control the pulse duty cycle and illumination time of multiple sets of sub-beams.

[0029] Optionally, the lens group includes an attenuation wheel, a beam expander, a replacement lens, and a shaping lens;

[0030] The attenuation wheel, the beam expander, and the replacement mirror are sequentially arranged between the source light source and the optical template, with the replacement mirror positioned close to the optical template and the attenuation wheel positioned close to the source light source.

[0031] The attenuation wheel is used to adjust the illumination intensity of the multiple sets of sub-beams;

[0032] The shaping lens is disposed between the optical template and the mechanical control platform. The shaping lens is used to parallelize and collimate the light reflected from the optical template and output multiple sets of sub-beams.

[0033] Optionally, the shaping lens includes a first locking lens, an interface lens, a second locking lens, and a spatial aperture.

[0034] The interface lens is positioned close to the optical template, the spatial aperture is positioned between the interface lens and the first locking lens, the first locking lens is positioned between the second locking lens and the spatial aperture, and the second locking lens is positioned close to the mechanical control platform.

[0035] Optionally, a light-shielding pad is provided at the bottom of the microporous plate, the light-shielding pad including an array of light-transmitting holes and an opaque area;

[0036] The light-blocking pad covers the portion of the microplate that does not have culture wells;

[0037] Each sub-beam penetrates only the corresponding light-transmitting hole on the light-shielding pad.

[0038] Optionally, the imaging device includes a fluorescence color wheel, an excitation light source, an imaging camera, an emission light filter, a dichroic mirror, and a fluorescence microscope lens;

[0039] The fluorescence microscope head is located close to the mechanical control platform. The fluorescence color wheel and the dichroic mirror are positioned between the excitation light source and the fluorescence microscope head. The dichroic mirror is tilted at 45 degrees toward the imaging camera. The emission filter is positioned between the dichroic mirror and the imaging camera.

[0040] Beneficial effects:

[0041] This application provides a multi-wavelength array-type light modulation and detection device for optogenetics. It comprises an array-type light source, a mechanical control platform, an imaging device, and a control module. The mechanical control platform houses a microplate with multiple arrayed culture wells for cell culture. The control module is connected to the array-type light source and includes a control circuit board containing a light source driver. The array-type light source emits multiple sets of sub-beams distributed in an array towards the microplate, with each set of sub-beams corresponding to different culture wells on the microplate. Each set of sub-beams has different target parameters, including wavelength, light intensity, pulse duty cycle, and duration. Because each set of sub-beams emits light with different target parameters, the illumination received by the culture wells at different positions on the microplate is not the same. Furthermore, the target parameters of the illumination received by each culture well can be adjusted as needed, thereby enabling experiments with multiple different parameters to be performed, resulting in higher experimental throughput and lower cost. This application discloses a multi-wavelength array-type light regulation and detection device for optogenetics, which can be used to construct a multi-wavelength array-type light regulation experimental platform with controllable light intensity and time, thereby exploring the potential of optogenetics in high-throughput metabolic regulation of cells. Attached Figure Description

[0042] Figure 1A This is a schematic diagram of the structure of an array light source for a multi-wavelength array-type light modulation and detection device for optogenetics, as proposed in one embodiment of this application.

[0043] Figure 1B A schematic diagram of a multi-wavelength array-type light modulation and detection device for optogenetics is provided in one embodiment of this application;

[0044] Figure 2 This is a schematic diagram of a set of sub-light sources for a multi-wavelength array-type light modulation and detection device for optogenetics according to an embodiment of this application;

[0045] Figure 3 This is a schematic diagram of the structure of a control circuit board for a multi-wavelength array-type light modulation and detection device for optogenetics, according to an embodiment of this application.

[0046] Figure 4 This is a schematic diagram illustrating the light modulation principle of a multi-wavelength array light modulation and detection device for optogenetics, as proposed in one embodiment of this application.

[0047] Figure 5 A is a schematic diagram of the structure of a light-shielding pad for a multi-wavelength array light modulation and detection device for optogenetics according to an embodiment of this application;

[0048] Figure 5 B is a schematic diagram of the structure of a light-shielding cover for a multi-wavelength array light modulation and detection device for optogenetics according to an embodiment of this application; Figure 5 C- Figure 5 D is a schematic diagram of the instrument housing of a multi-wavelength array light modulation and detection device for optogenetics according to an embodiment of this application;

[0049] Figure 6 This is a software flowchart of a multi-wavelength array light modulation and detection device for optogenetics proposed in one embodiment of this application;

[0050] Figure 7 This is a schematic diagram of the overall structure of another multi-wavelength array-type light modulation and detection device for optogenetics proposed in an embodiment of this application;

[0051] Figure 8 This is a schematic diagram of the optical template and lens group of another multi-wavelength array-type light modulation and detection device for optogenetics proposed in one embodiment of this application;

[0052] Figure 9 This is a schematic diagram of the light modulation principle of another multi-wavelength array light modulation and detection device for optogenetics proposed in an embodiment of this application;

[0053] Figure 10 This is a schematic diagram of the source light source of another multi-wavelength array light modulation and detection device for optogenetics proposed in an embodiment of this application;

[0054] Figure 11 A- Figure 11 B is a graph showing the wavelength range and measured values ​​of a multi-wavelength array-type optical modulation and detection device for optogenetic proteins proposed in an embodiment of this application.

[0055] Figure 12 A is a schematic diagram of a medium-throughput microplate for a multi-wavelength array-type light modulation and detection device for optogenetic proteins, according to an embodiment of this application.

[0056] Figure 12 B is a top view of a high-throughput microplate for a multi-wavelength array-type light modulation and detection device for optogenetic proteins, as proposed in an embodiment of this application.

[0057] Figure 12 C is a side view of a high-throughput microplate for a multi-wavelength array-type light modulation and detection device for optogenetic proteins, according to an embodiment of this application.

[0058] Figure labeling: 1. Microplate; 10. Medium-throughput microplate; 101. Surrounding area of ​​well; 102. Bottom well; 111. Light-shielding layer; 112. Light-transmitting hole; 115. Cell culture well; 2. Mechanical control platform; 3. Lens group; 31. Attenuation wheel; 32. Beam expander lens; 35. Replacement lens; 33. Shaping lens; 310. Electrically controlled rotating wheel; 311. Continuous attenuation plate; 321. First beam expander lens; 322. Second beam expander lens; 331. First multiplier lens; 332. Interface lens; 333. Second multiplier lens; 334. Spatial aperture; 4. Optical template; 41. Digital mirror; 5. Light source; 50. Sub-light source; 51. Condenser lens; 52. Light source array; 54. Source light source; 55. Light-emitting diode; 61. Control circuit 62. Light source driver; 621. First light source driver; 622. Second light source driver; 623. Third light source driver; 605. Socket; 606. Power chip; 608. Driver chip; 607. Resistor-capacitor components; 609. External DC power supply; 610. Array substrate; 69. Microcontroller; 68. Light-shielding pad; 681. Transparent area; 682. Opaque area; 7. Imaging device; 71. Fluorescent color wheel; 72. Excitation light source; 73. Imaging camera; 74. Emitting light filter; 75. Dichroic mirror; 76. Fluorescence microscope lens; 80. Instrument housing; 801. Window on the top cover of the housing; 85. Microplate light-shielding cover; 851. Micro-hole interlocking pillar; 852. Top cover plane; 853. Handle. Detailed Implementation

[0059] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0060] Reference Figure 1B As shown in the embodiment of this application, a multi-wavelength array light modulation and detection device for optogenetics is disclosed. The device includes an array light source 5, a mechanical control platform 2, an imaging device 7, and a control module.

[0061] Specifically, the array light source 5 is set on one side of the mechanical control platform 2, and the imaging device 7 is set on the side of the mechanical control platform 2 away from the light source 5. The array light source 5, the mechanical control platform 2 and the imaging device 7 are on the same straight line. It can be understood that when using the device, the array light source 5 is located below the mechanical control platform 2 and the imaging device 7 is located above the mechanical control platform 2. Thus, the array light source 5 emits light from bottom to top.

[0062] A microplate 1, comprising multiple culture wells for cell culture, is mounted on the mechanical control platform 2. The mechanical control platform 2 is used for the precise movement and positioning of the microplate 1, as well as for oscillation and mixing during photoadjustment experiments; it also supports the microplate 1 and moves it precisely along the horizontal X and Y axes, assisting the positioning and calibration of the imaging device 7 during fluorescence imaging. In this embodiment, the mechanical control platform 2 employs a U-780 motorized stage with a displacement accuracy within 10 μm.

[0063] Microplate 1 is used for cell culture, including but not limited to various forms of microreactors such as microplates and micro-pit array chips. The external dimensions of the microplate can be 128mm*96mm to accommodate large-scale parameter combination screening.

[0064] For example, the microplate 1 can be a medium-throughput biological experimental microplate, including 96 wells, 384 wells, and 1536 wells. In an optional embodiment, a microplate with transparent bottom wells 102 and opaque surrounding area 101 can be selected, such as Corning 3340 / 3603 / 3416 black transparent bottom microplates.

[0065] For example, refer to Figure 12 A- Figure 12As shown in Figure C, the microplate 1 can also be a higher-throughput microwell array chip, such as a microwell array chip with a well spacing of 0.5 mm, a well diameter of 0.5 mm, and more than 10,000 wells. The microwell array chip includes an opaque light-shielding layer 111 and cell culture wells 115 for cell culture, with transparent light-transmitting holes 112 at the bottom of the cell culture wells. Different inner diameters and periodic spacings of microwells / wells can be designed according to the different cells to be cultured. It should be noted that the optogenetic protein genes regulated by the cells in each well or well can be combined, and the optics between the wells are separated by the light-shielding layer and do not affect each other.

[0066] In this application, the parameters of the microplate 1 can be controlled by combining the wavelength selection, gradient light intensity control, and light irradiation time of the array light source 5.

[0067] Furthermore, the control module is connected to the array light source 5. The control module includes a control circuit board 61, which includes a light source driver 62. The light source driver 62 is used to make the array light source 5 output multiple sets of sub-beams distributed in an array, and the positions of the multiple sets of sub-beams correspond to the culture wells at different positions on the microplate 1.

[0068] Specifically, refer to Figure 1B As shown, the control circuit board 61 also includes an array substrate 610. The array-type light source 5 can be mounted in a corresponding array on the array substrate 610, thereby emitting multiple sets of sub-beams distributed in an array. The sub-beams are emitted from the array-type light source 5 toward the microplate 1. Furthermore, the position of each set of sub-beams corresponds to several culture wells at different positions on the microplate 1, and the light emitted by each set of sub-beams has different target parameters. That is, the light from each set of sub-beams will only illuminate several culture wells along its light path, without affecting other surrounding culture wells, and there will be no mutual interference.

[0069] Furthermore, the aforementioned culture wells can be a single well or an integer square (e.g., 4, 9) of culture wells, all sharing the same light irradiation experimental conditions. Further, refer to... Figure 2 As shown, the array light source 5 includes multiple sets of sub-light sources 50, and each set of sub-light sources 50 can generate a corresponding sub-beam.

[0070] Specifically, the sub-light source 50 includes multiple micro light-emitting diodes, and the light emitted by the multiple micro light-emitting diodes in each group of sub-light sources has different wavelengths.

[0071] Furthermore, referring to Figure 3 and Figure 4 As shown, the control circuit board 61 also includes multiple light source drivers 62, a power chip 606, a microcontroller 69, a socket 605, a driver chip 608, resistive and capacitive components 607, and an external DC power supply 609.

[0072] Specifically, multiple light source drivers 62 correspond to and are connected to multiple sub-light sources 50. The multiple light source drivers 62 are used to drive the corresponding sub-light source 50 to emit corresponding sub-beams toward the microporous plate 1. The light source drivers 62 can make the light emitted by each sub-light source 50 have different target parameters. For example, the target parameters may include light wavelength, light intensity, pulse duty cycle, and illumination time, etc., so that multiple experimental groups with different light control parameters can be set in the experiment.

[0073] Reference Figure 4 As shown, the control circuit board 61 can adjust the target parameters of the sub-beams emitted by different sub-light sources 50 by independently adjusting the driving current of different sub-light sources at different times. Specifically, the voltage regulator chip 606 in the control circuit board 61 receives the VCC of the external DC power supply 609 via a wired connection and provides a stable voltage to each unit of the control circuit board 61. The microcontroller 69 drives multiple light source drivers 62 in series via a bus through the socket 605. Each light source driver 62 is connected to the cathode of the corresponding sub-light source 50, that is, connected to the output pin of the light source driver 62; the anode of each sub-light source 50 is connected to the voltage regulator chip 606, and each light source driver 62 includes a decoupling capacitor.

[0074] Reference Figure 3 As shown, the microcontroller 69 is connected to multiple light source drivers 62. The microcontroller 69 can send light modulation commands to the multiple light source drivers 62, and the light source drivers 62 can respond to the light modulation commands to independently adjust the illumination intensity, pulse duty cycle, and illumination time of the corresponding sub-light source. For example, the light source drivers 62 can realize the independent driving of a high-density array light source. For instance, the array light source 5 includes a total of 288 micro-light-emitting diodes, which are divided into different control groups according to the differences in the required light wavelength. Specifically, the first group of light source drivers 621 can drive 96 sub-light sources 50 corresponding to the first wavelength, the second group of light source drivers 622 can drive 96 sub-light sources 50 corresponding to the second wavelength, and the third group of light source drivers 623 can drive 96 sub-light sources 50 corresponding to the third wavelength. For example, the first wavelength is 455nm, the second wavelength is 630nm, and the third wavelength is 654nm.

[0075] Furthermore, referring to Figure 1BAs shown, the imaging device 7 is used for in-situ detection of cells on a microplate. The imaging device includes a fluorescence color wheel 71, an excitation light source 72, an imaging camera 73, an emission light filter 74, a dichroic mirror 75, and a fluorescence microscope head 76; wherein, the fluorescence microscope head 76 is close to the mechanical control platform 2, the fluorescence color wheel 71 and the dichroic mirror 75 are disposed between the excitation light source 72 and the fluorescence microscope head 76, and the dichroic mirror 75 is tilted towards the imaging camera 73, and the emission light filter 74 is disposed between the dichroic mirror 75 and the imaging camera 73.

[0076] Specifically, the fluorescence microscope lens 76 is used for imaging focusing, the fluorescence color wheel 71, the excitation light source 72, and the emission light filter 74 are used to select the required illumination, excitation, or emission light wavelengths, and the imaging camera 73 is used to capture cell status. This application adopts a spatial separation scheme for bottom illumination and top detection of the microplate 1, with the imaging light source 72 and the array light source 5 in a time-division / wavelength-division multiplexing form. Furthermore, the excitation light source 72 has a wavelength close to 350nm, the emission light filter 74 uses a wavelength close to 700nm, and the array light source 5 has wavelengths of 400-650nm located in different bands, which can be separated by the fluorescence color wheel 71 and the emission light filter 74; in addition, the array light source 5 and the excitation light source 72 can also be time-division multiplexed. That is, the imaging light source does not affect the light control system, and is compatible with image and fluorescence detection of the status of strains in each well / pit, allowing for in-situ simultaneous light control and cell detection.

[0077] The multi-wavelength array light modulation and detection device for optogenetics provided in this application embodiment uses multiple light source drivers 62 to drive multiple sub-light sources 50 to emit corresponding sub-beams toward the microplate 1. Since the light of each group of sub-beams has different target parameters, the illumination received by the culture wells at different positions on the microplate 1 is different, and the illumination received by each culture well can be adjusted according to the needs, thereby completing multiple sets of experiments with different parameters, resulting in higher experimental throughput and lower cost.

[0078] Meanwhile, the multi-wavelength array-type light regulation and detection device for optogenetic proteins provided in this application embodiment makes full use of the bottom space of the microplate 1 to perform multi-wavelength independent light regulation at different positions. Simultaneously, the top space of the microplate 1 can be used in conjunction with devices such as an inverted fluorescence microscope and a microplate reader. This achieves bottom light regulation while simultaneously detecting the culture and fermentation parameters of the microplate 1.

[0079] Furthermore, the multi-wavelength array-based optical modulation and detection device for optogenetic proteins provided in this application can meet the current research needs for multi-parameter optical modulation and detection of optically controlled genes / opogenetic proteins with different functions. It can be used to construct a multi-wavelength array-based optical modulation experimental platform with controllable light intensity and time, thereby exploring the potential of optogenetics in high-throughput metabolic regulation of cells. The device achieves optimal optical control parameters through micro-system screening and effectively avoids the drawbacks of chemically induced regulation methods. It possesses numerous advantages such as being non-destructive, non-invasive, having high spatiotemporal resolution, and being highly reversible.

[0080] In one alternative implementation, the light source driver 62 employs an industrial LED driver TLC59414 and operates in a daisy-chain configuration. In this application, each TLC5941 can control 24 LEDs and independently adjust their drive current; that is, one light source driver 62 controls 24 light channels and operates in a constant current state.

[0081] In one alternative implementation, refer to Figure 1A and Figure 2 As shown, illumination of different wavelengths is achieved by multiple side-by-side light source arrays 52. Individual sub-light sources (containing LEDs of different wavelengths) can be placed at the bottom of several culture wells to achieve simultaneous excitation and control of multiple wavelengths. For example, an array 52 containing 96 different optical wavelength combinations is designed for a 96-well / 384-well plate. Each sub-light source 50 in these 96 channels can be controlled independently. Therefore, the optical screening conditions can be different for each well in a 96-well plate, one optical screening condition is shared for every four wells in a 384-well plate, and approximately 100 wells share one optical screening condition for a 10,000 microarray chip. A total of 288 independent LED light sources are driven by 12 light source drivers 62. Each LED can generate a 24-channel, linearly adjustable current with a maximum current of 30mA and 12-bit resolution intensity (i.e., 4096 different light intensities).

[0082] In one alternative implementation, the device can use an SPI bus to drive multiple light source drivers 62 in series, and be controlled by the controller port of the control module. Current control data and PWM control data can be written from the SIN input terminal, allowing the source drivers 62 to generate pulses with different PWM duty cycles to achieve pulse ratio adjustment in the target parameters. The PWM reference clock can be provided by an internal oscillator. The multiple light source drivers 62 implement a constant current receiver driver, controlled via a serial interface consisting of pins BLANK (2), XLAT (3), SCLK (4), SIN (5), MODE (6), and GSCLK (25). The serial connection of the multiple light source drivers 62 is achieved by connecting the SIN (5) of the next light source driver 62 to the SOUT (24) pin of the previous light source driver 62; the SOUT pin of the last driver does not need to be connected; the IREF (27) pin is grounded through a 2.2kΩ resistor. In this embodiment, the measured maximum current of each output of the light source driver 50 is 17.8mA.

[0083] In one alternative implementation, the control module is controlled by an ATmega32u4 microcontroller. During device operation, this controller stores and executes user-defined optical modulation configuration files, eliminating the need for an external computer and providing power via a 606 power management system. The ATmega32u4 is an 8-bit AVR RISC microcontroller equipped with 32KB flash memory, 2.5KB SRAM, 1KB EEPROM, a 10-bit analog-to-digital converter, and a JTAG interface for on-chip debugging. This control module includes the control functions required to support multi-wavelength array optical modulation and can operate independently. If optical modulation parameters need to be modified, simply connecting to a computer via USB cable allows for restarting the software configuration of the multi-wavelength array optical modulation device.

[0084] In one alternative implementation, considering that the heat generated by long-term illumination of the light source (affecting the environment around the light source and the environment within the cell culture medium) may affect cell growth, a thermal management system is designed to maintain a suitable environment for cell growth in the microplate. In this embodiment, thermally conductive silicone grease, a thermally conductive silicone pad, a thermally conductive aluminum plate, a heat sink, and a temperature-controlled fan are sequentially added to the bottom of the control circuit board 61, and the entire assembly is mounted on a single substrate.

[0085] The driver chip 608 on the control circuit board 61 can be used to regulate the speed of the temperature-controlled fan and the temperature of the bottom temperature control unit. For example, the bottom temperature control unit uses a thermal resistance heating method and is located inside the heat-conducting aluminum plate, which can control the temperature at the bottom of the control circuit board 61. If a heat-dissipating zone is required, the heat-conducting aluminum plate can be divided. When power is applied for a light control experiment, the software program ensures that the light source illuminates according to the program settings, the fan operates at the speed set in the program, and the temperature inside the microplate is measured. The fan speed and the temperature of the heat-conducting aluminum plate are adjusted to bring the solution inside the microplate to the set temperature value.

[0086] In one alternative implementation, to ensure optical isolation between different culture wells, a light-shielding pad 68 is designed, for example, a laser-cut nitrile sheet pad, to prevent light leakage and crosstalk between culture wells. Figure 5 A shows the gasket for a 96-well plate; the light-shielding gasket design for other perforated plates such as 384-well / 1536-well plates is similar.

[0087] The light-shielding pad 68 includes an array of light-transmitting holes 681 and an opaque area 682. The opaque area 682 covers the portion of the microplate not containing culture wells, and each sub-beam penetrates only the corresponding light-transmitting hole on the light-shielding pad. The top of the light-shielding pad 68 has a rectangular recessed groove for securing the placed object. Preferably, the light-shielding pad 68 can be 128mm x 96mm in size to accommodate and secure various microplates, such as Corning 3340 / 3603 / 3416 black transparent-bottomed microplates.

[0088] In one optional implementation, the sub-light sources of different wavelengths are preferably monochromatic LED beads of different bands, each LED bead corresponding to a wavelength-specific optogenetic protein. In the embodiments of this application, the monochromatic LEDs have strong wavelength selectivity, covering the regulation wavelengths of six major classes of optogenetic proteins in the ultraviolet-visible light range (300-700nm), and can be specifically and differentially selected according to the wavelength required for the excitation or reversal of optogenetic proteins, thus it can be used for light regulation involving different wavelengths. In the embodiments of this application, the monochromaticity of LEDs in some wavelength bands is poor, with a full width at half maximum (FWHM) greater than 30nm, which affects precise light control; the power of monochromatic LEDs is low at certain wavelengths, such as 480nm and 640nm, requiring careful selection of LED devices based on experimental requirements, cost, and availability.

[0089] In an optional implementation, this application embodiment also includes a device housing 80. For example... Figure 5 B- Figure 5D, wherein the upper cover 801 of the instrument housing has a rectangular opening corresponding to the array substrate 610, for placing the microporous plate 1. The entire system is integrated and fixed to the one-piece housing 8, increasing its resistance to long-period, large-amplitude vibration interference. A light-shielding cover 85 is provided, which consists of a handle 853, a microporous fitting post 851, and an opaque upper cover plane 852, for securely covering the microporous plate, and is fitted into the transparent bottom hole by the microporous fitting post 851. Furthermore, the above device can be fixed in a shaker, using the circular motion of the shaker for oscillation, and avoiding the problem of loosening and falling off of mechanical fixing structures such as heat sink components and fixing components due to long-term vibration.

[0090] The device disclosed in this application only requires setting the internal program code and connecting a corresponding external power adapter to achieve the predetermined optogenetic protein regulation function. For example... Figure 6 The diagram shown is a software flowchart for a multi-wavelength array-based optical modulation and detection device for optogenetic proteins, including the following steps:

[0091] 1. Define the light source control parameters for a multi-wavelength array, such as a light control parameter matrix for 96*3 LEDs. The parameters should include at least: the wavelength of the illuminated light, the light intensity, the illumination interval, and the total illumination duration.

[0092] 2. Initialize the baud rate, the state of each sub-light source 50, the phase time, the temperature of the microplate 1, and the fan speed;

[0093] 3. Set the light control to constant lighting or pulsed lighting mode;

[0094] 4. Based on the set parameter matrix, determine whether to activate delayed lighting;

[0095] 5. If the light-on time requirement is met, turn on the sub-light source 50 at the corresponding position of the micro-perforated plate, enter the light source control state of the multi-wavelength array; and continuously read the target parameters to complete the set illumination parameters in the light control parameter matrix.

[0096] 6. Once the total illumination time is reached, the system will automatically shut down after illumination ends.

[0097] After constructing cells regulated by different optogenetic proteins, they can be transferred to the above-mentioned medium-throughput / high-throughput microplates for culture, and the parameters of optogenetic proteins studied in the embodiments of this application can be detected by different light parameters.

[0098] In one alternative implementation, refer to Figure 7 As shown in the embodiments of this application, a multi-wavelength array light modulation and detection device for optogenetics is also disclosed. In this device, the array light source 5 includes a source light source 54, an optical template 4, and a lens group 3.

[0099] Specifically, refer to Figure 7 As shown, the optical template 4 is disposed on one side of the mechanical control platform 2, the imaging device 7 is disposed on the side of the mechanical control platform 2 away from the optical template 4, and the optical template 4, the mechanical control platform 2 and the imaging device 7 are on the same straight line. The source light source 54 is located on one side of the optical template 4, and the source light source 54 and the mechanical control platform 2 are not on the same straight line. The lens group 3 is disposed between the source light source 54 and the optical template 4.

[0100] Furthermore, the control module is connected to the optical template 4. The control module is used to send adjustment commands to the optical template 4. The optical template 4 is used to regulate the original beam emitted by the source light source according to the adjustment commands, and generate multiple sets of sub-beams with target parameters, and project the regulated sub-beams toward the mechanical control platform. The light source driver 62 is connected to the source light source 54 and is used to drive the source light source 54 to emit original beams of multiple wavelengths, and regulate the wavelength of the original beam emitted by the source light source 54.

[0101] Specifically, refer to Figure 8 As shown, the optical template 4 includes a digital mirror (DMD) 41. The digital mirror 41 is disposed on the side of the mechanical control platform 2 away from the imaging device 7, and the digital mirror 41 is tilted.

[0102] The digital reflector 41 is connected to the control module. The digital reflector 41 is used to control the digital reflector 41 to addressably adjust the reflection of the original light beam from the source light source 54 according to the adjustment instructions of the control module, and to project multiple sets of sub-beams distributed in an array.

[0103] Furthermore, in this embodiment, the optical template 4 employs a digital reflector 41 (DMD). This device utilizes multiple 10μm-20μm micromirrors to addressably select optical wavelengths at different locations on the microperforated plate 1. By controlling the underlying CMOS control circuitry of the digital reflector 41, each micromirror can be individually controlled, allowing each micromirror to spatially select (open) or deviate (close) from the original light beam from the source light source 54, creating either bright or dark pixels at the bottom of the microperforated plate 1, thus forming multiple sets of sub-beams. The optical template 4, composed of a high-speed digital optical reflection switch array, has a very fast response time, approximately in the microsecond range from fully on to fully off, making it suitable as a spatial light modulator. Combined with the source light source 54, it can "digitally" achieve parameter adjustment of light modulation. Furthermore, the digital reflector 41 can control the pulse duty cycle and illumination time of the multiple sets of sub-beams.

[0104] Furthermore, the source light source 54 employs multiple monochromatic light sources that meet the wavelength requirements of biological experiments, and by changing the wavelength of the monochromatic LED beads, at least three different wavelength light sources can be freely combined. Specifically, refer to... Figure 10 As shown, the light source 54 may include multiple light-emitting diodes 55 with different wavelengths and a condenser lens 51. The light-emitting diodes 55 are fixed at a certain distance from the center of the condenser lens 51. The condenser lens 51 can focus the light from the light-emitting diodes 55 to the lens group 3, and then the lens group 3 and the optical template 4 perform array-type light control.

[0105] Furthermore, referring to Figure 7 and Figure 8 As shown, the lens group 3 includes an attenuation wheel 31, a beam expander 32, a replacement lens 35, and a shaping lens 33. The attenuation wheel 31, the beam expander 32, and the replacement lens 35 are sequentially arranged between the light source 5 and the optical template 4, with the attenuation wheel 31 positioned closer to the light source 5 and the replacement lens 35 positioned closer to the optical template 4. The shaping lens 33 is arranged between the optical template 4 and the mechanical control platform 2, and is used to collimate and parallelize the light rays projected from the optical template 4.

[0106] Specifically, the attenuation wheel 31 includes an electrically controlled rotating wheel 310 and a continuous attenuator 311. The attenuation wheel 31 is used to control the illumination intensity of multiple sets of sub-beams. The beam expander lens 32 includes a first beam expander 321 and a second beam expander 322, used to expand the diverging point light source of the source light source 54 to the size of the digital reflector array 41. The successor lens 35 is used for optical path succession, and in some embodiments, the successor lens 35 may not be provided.

[0107] Reference Figure 7 and Figure 8 As shown, the shaping lens 33 includes an interface lens 332, a spatial aperture 334, a first locking lens 331, and a second locking lens 333. The interface lens 332 is positioned close to the optical template 4, the spatial aperture 334 is positioned between the interface lens 332 and the first locking lens 331, the first locking lens 331 is positioned between the second locking lens 333 and the spatial aperture 334, and the second locking lens 333 is positioned close to the mechanical control platform 2.

[0108] Reference Figure 9 As shown, the optical template 4 in this application is driven by adjustment commands from the control module, and its design block diagram is as follows. Figure 4As shown. The control module includes a control circuit board 61, a power manager 62, a digital mirror controller 63, and a microcontroller 69. The microcontroller 69 receives and processes control signals, then sends adjustment commands to the digital mirror controller 63, which directly performs addressing and gating of the optical template 4. The power manager 62 provides the overall DC power supply. The digital mirror controller 63 regulates the optical template 4 by controlling the deflection data of the digital mirror 41, as well as the overall power supply 46 and optomechanical components of the optical template 4. These optomechanical components include, but are not limited to, replacement mirrors and folding mirror assemblies. At this time, the control circuit board 61 controls the gating of different wavelengths of light from the source light source 54.

[0109] It should be noted that the digital reflector device in this application can be developed using a commercial DMD original equipment manufacturer (OEM) kit, and the entire optical system can be designed independently based on the performance parameters of the light source. Alternatively, an optomechanical component integrating a DMD kit can be used, and the source light source 54, lens group 3, and optical template 4 can be designed independently according to actual needs. In this embodiment, the optomechanical component of the digital micromirror DMD is used, preferably the PRO4500VIS700 optomechanical component, which integrates a 1980*1080 micromirror array. The micromirrors are all suspended and can be tilted to both sides by about 10-12°, thereby forming two working states: light on and light off. Due to the high density and small size of the digital reflector device, high-density array-type light control can be performed. For example, 1980*1080 light states can be controlled within a 128mm x 96mm space, and at least three different wavelengths of light intensity and illumination time can be independently controlled simultaneously within a local microspace (0.1-3mm), meeting the needs of different types of multi-parameter light control and detection.

[0110] The multi-wavelength array light modulation and detection device for optogenetics provided in Embodiment 2 of this application combines illumination parameters at different locations using an addressable optical template 4. The sub-beams emitted by the source light source 54, expanded by the lens group 3, and modulated by the optical template 4 are shaped and adjusted by a shaping lens, and finally irradiated onto the microplate 1. This enables independent adjustment of the wavelength, intensity, and time of the irradiation light for each culture well of the microplate, thereby completing experiments with multiple sets of different parameters, resulting in higher experimental throughput and reduced costs.

[0111] To verify the application effect of the multi-wavelength array-based photoregulation and detection device for optogenetic proteins in this application, this application also experimentally verified the photoregulation of the single-component blue light-induced protein EL222. The EL222 transcription factor, originally derived from a bacterial photo-oxygen-pressure protein, dimers and binds to DNA upon blue light irradiation. This system has a sufficiently large tunable expression range for the target protein, can rapidly activate and deactivate, and exhibits a sufficiently high linear correlation with light intensity. The light-dependent transcriptional activation of the EL222 system requires only a few elements: a photosensitive domain LOVII and a helical-turn-helical DNA-binding domain. In the dark, the LOVII domain binds to the HTH domain, covering the HTH4α site necessary for dimerization and binding to DNA. Blue light irradiation triggers photochemical reactions: the flavoprotein complex in the LOV domain disrupts the interaction between LOV and HTH, causing EL222 to dimerize and thus exert its DNA-binding protein activity. These reactions will then proceed in reverse in black iris, and the reverse reaction will occur quickly after blue light irradiation is stopped.

[0112] The multi-wavelength array-type optical modulation and detection device described in this application can typically use light source spectral wavelengths such as... Figure 11 As shown in Figure A, in one example embodiment, the light intensity and spectral distribution of different micropores in the device were measured, and the results are as follows: Figure 11 As shown in B, the center wavelength and intensity results of repeated measurements are shown in Table 1, verifying the effectiveness of the device and its applicability for parameter detection and optimization experiments.

[0113] Table 1

[0114]

[0115]

[0116] This application adopts a multi-wavelength array addressable light control scheme. The overall device can realize the independent simultaneous control of light intensity and illumination time of multiple different wavelengths within a local microspace (0.1-3mm), which meets the needs of different types of optogenetic proteins for multi-parameter light control and detection.

[0117] This application is also compatible with various forms of microplates, such as microplates and micro-pit array chips. The first type of microplate is a medium-throughput microplate (including 96, 384, and 1536 wells), and the second type of microplate is a higher-throughput micro-pit array chip (>10,000 wells), which has higher experimental throughput and is compatible with a larger-scale parameter combination screening.

[0118] This application adopts a spatial separation scheme for bottom illumination and top compatible detector of microplate. The imaging light source and controllable light source can also be reused in time-division / wavelength division, that is, the imaging light source does not affect the light control system. With the help of light shielding pads, light control and detection can be performed simultaneously in situ. It also takes into account the stability and controllability of the cell culture environment, and multiple parameters such as temperature can be fed back and controlled in real time.

[0119] This application presents a multi-wavelength array-based light modulation and detection device for optogenetics. It fully utilizes the bottom space of a microplate to perform multi-wavelength array-based light modulation at different locations, enabling the construction of a multi-wavelength array-based light modulation experimental platform with controllable light intensity and time. This allows for the exploration of optogenetics' potential in high-throughput metabolic regulation of cells. Furthermore, it provides a solution that simultaneously meets the needs of light modulation and real-time detection. The innovative advantages of utilizing a light-controlled switch for multi-gene research allow for screening and optimization experiments with different parameters to be performed in the same batch under high-throughput light modulation conditions, improving experimental throughput and reproducibility while reducing errors.

[0120] It should be noted that the various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0121] It should also be noted that, in this document, the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. Furthermore, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations, nor should they be construed as indicating or implying relative importance. Moreover, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. In the absence of further restrictions, an element defined by the phrase "includes a..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes the element.

[0122] The technical solutions provided in this application have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand this application, and the content of this specification should not be construed as a limitation of this application. Furthermore, for those skilled in the art, there will be different forms of changes in the specific implementation methods and application scope based on this application. It is neither necessary nor possible to exhaustively list all implementation methods here, and obvious changes or modifications derived therefrom are still within the protection scope of this application.

Claims

1. A multi-wavelength array-type light modulation and detection device for optogenetics, characterized in that, include: Array-type light source, mechanical control platform, imaging device and control module; The array-type light source is located on one side of the mechanical control platform, and the imaging device is located on the side of the mechanical control platform away from the light source. The array-type light source, the mechanical control platform, and the imaging device are on the same straight line. The mechanical control platform is equipped with a microplate, which includes multiple culture wells for cell culture. The control module is connected to the array light source, and the control module includes a control circuit board, which includes a light source driver. The array-type light source outputs multiple sets of sub-beams distributed in an array, and the positions of the multiple sets of sub-beams correspond to the culture wells at different positions on the microplate. Each group of sub-beams has different target parameters, and the sub-beams are emitted from the array of light sources toward the microporous plate; The imaging device is used to perform in situ detection of cells cultured on the microplate; The target parameters include light wavelength, light intensity, pulse duty cycle, and illumination time; The array-type light source includes multiple sets of sub-light sources, and multiple light source drivers are provided, with each set of light source drivers connected to multiple sets of sub-light sources respectively. The control circuit board also includes a microcontroller, which is connected to a plurality of the light source drivers and is used to send light modulation commands to the light source drivers. The light source driver responds to the light modulation command by selecting and turning on the sub-light source of the target wavelength, driving the sub-light source to generate the corresponding sub-beam, and adjusting the light intensity, pulse duty cycle and illumination time of the sub-light source according to the light modulation command. The bottom of the microporous plate is provided with a light-shielding pad, which includes an array of light-transmitting holes and an opaque area. The opaque area covers the portion of the microplate where no culture wells are provided; Each sub-beam penetrates only the corresponding light-transmitting hole on the light-blocking pad.

2. The multi-wavelength array-type light modulation and detection device for optogenetics according to claim 1, characterized in that: The sub-light source includes multiple micro light-emitting diodes, and the wavelengths of light emitted by the multiple micro light-emitting diodes in each group of sub-light sources are different.

3. The multi-wavelength array-type light modulation and detection device for optogenetics according to claim 1, characterized in that: The array-type light source includes a source light source, an optical template, and a lens group; The optical template, the mechanical control platform, and the imaging device are on the same straight line, the source light source is located to the side of the optical template, and the lens group is disposed between the source light source and the optical template. The control module is connected to the optical template, and the control module is used to send adjustment commands to the optical template; The optical template is used to regulate the original beam emitted by the source light source according to the adjustment command, generate multiple sets of sub-beams with target parameters, and project the regulated sub-beams toward the mechanical control platform. The light source driver is connected to the source light source and is used to drive the source light source to emit a raw light beam and to adjust the wavelength of the raw light emitted by the source light source.

4. The multi-wavelength array-type light modulation and detection device for optogenetics according to claim 3, characterized in that: The optical template includes a digital reflector, which is disposed on the side of the mechanical control platform away from the imaging device, and the digital reflector is tilted. The light source includes multiple light-emitting diodes with different wavelengths and a condenser lens, and the condenser lens focuses the light from the light-emitting diodes onto the lens group; The digital reflector is connected to the control module. The digital reflector is used to control the addressable adjustment and reflection of the original light from the source light source according to the adjustment command of the control module, and to project multiple sets of sub-beams distributed in an array. The digital reflector is used to control the pulse duty cycle and illumination time of multiple sets of sub-beams.

5. The multi-wavelength array-type light modulation and detection device for optogenetics according to claim 3, characterized in that: The lens group includes an attenuation wheel, a beam expander, a replacement lens, and a shaping lens; The attenuation wheel, the beam expander, and the replacement mirror are sequentially arranged between the source light source and the optical template, with the replacement mirror positioned close to the optical template and the attenuation wheel positioned close to the source light source. The attenuation wheel is used to adjust the illumination intensity of the multiple sets of sub-beams; The shaping lens is disposed between the optical template and the mechanical control platform. The shaping lens is used to parallelize and collimate the light reflected from the optical template and output multiple sets of sub-beams.

6. The multi-wavelength array-type light modulation and detection device for optogenetics according to claim 5, characterized in that: The shaping lens includes a first-magnification locking lens, an interface lens, a second-magnification locking lens, and a spatial aperture. The interface lens is positioned close to the optical template, the spatial aperture is positioned between the interface lens and the first locking lens, the first locking lens is positioned between the second locking lens and the spatial aperture, and the second locking lens is positioned close to the mechanical control platform.

7. The multi-wavelength array-type light modulation and detection device for optogenetics according to any one of claims 1-6, characterized in that: The imaging device includes a fluorescence color wheel, an excitation light source, an imaging camera, an emission light filter, a dichroic mirror, and a fluorescence microscope lens; The fluorescence microscope head is located close to the mechanical control platform. The fluorescence color wheel and the dichroic mirror are positioned between the excitation light source and the fluorescence microscope head. The dichroic mirror is tilted at 45 degrees toward the imaging camera. The emission filter is positioned between the dichroic mirror and the imaging camera.

Images