Multi-applicability real-time ratio light imaging device

Abstract

The invention provides a multi-applicability real-time ratio light imaging device. The multi-applicability real-time ratio light imaging device comprises a host, a time schedule controller, a light source, an image acquisition unit, a prism imaging light path, a light filter rotating wheel and an all-in-one illumination light path, the time sequence controller receives a time sequence control command sent by the host and sends high-frequency time sequence signals to the image acquisition unit and the light source respectively so as to control on-off of exciting light and acquisition of fluorescent light; the light sources are used for providing exciting light for exciting the fluorescent medicine to generate fluorescent light, the all-in-one illumination light path is used for connecting the multiple light sources into the exciting illumination light path at the same time, and the light filter rotating wheel switches corresponding light filters according to different light sources and is used for stopping interference of the exciting light on fluorescent imaging of the image acquisition unit; the prism imaging light path is used for collecting fluorescence and reflecting and transmitting the fluorescence to the corresponding image collection units according to different spectrums. By means of high integration, multi-functionalization and efficient fluorescence imaging capacity, filtering switching and experiment item switching can be carried out more quickly.

Description

Technical Field

[0001] The invention relates to the technical field of ratiometric optical imaging, and in particular to a multi-applicability real-time ratiometric optical imaging device. Background Art

[0002] Fluorescence, also known as "fluorescence", refers to a photoluminescent cold luminescence phenomenon, which generally includes several parameters or physical characteristics such as excitation spectrum, emission spectrum, fluorescence intensity, fluorescence quantum yield, fluorescence lifetime, and Stokes shift. The principle of fluorescence generation is: when light irradiates certain atoms, the energy of the light causes some electrons around the nucleus to jump from their original orbits to orbits with higher energy, that is, from the ground state to the first excited singlet state or the second excited singlet state. The first excited singlet state or the second excited singlet state is unstable, so it will return to the ground state. When the electron returns to the ground state from the first excited singlet state, the energy will be released in the form of light, so fluorescence is generated.

[0003] Fluorescence technology has a wide range of applications in many fields. Through fluorescence technology, people can sequence DNA, trace lymph nodes and tumors, detect drug metabolism, detect pesticide residues, etc. Usually, people use fluorescence technology mainly to collect fluorescence for fluorescence development to trace drugs, tumors, etc. With the development of technology, more and more fluorescence application methods have emerged.

[0004] Ratio imaging is a special fluorescence imaging technology, which is usually implemented in two ways: one is to alternately excite the fluorescent drug with two independent wavelengths of light and detect fluorescence at a single wavelength; the other is to excite the fluorescent drug with a single wavelength of light and detect the emission shift at two independent wavelengths. By comparing the fluorescence intensity collected twice, additional information can be obtained compared to a single fluorescence collection, such as changes in calcium ion concentration in the cell fluid, such as changes in the acid-base environment of the fluorescent drug solution.

[0005] like Figure 1 As shown, conventional ratiometric light imaging methods use a filter wheel to switch filters, and realize two independent wavelengths of excitation light by switching the filter of the excitation light source, or collect fluorescence at two independent wavelengths by switching the filter on the camera end. This method requires a motor to drive the filter wheel to repeatedly switch at high speed. Its switching speed is limited by the motor torque and speed of the filter wheel and the structural strength and stiffness of the filter wheel, and it is often impossible to switch quickly to achieve "real-time" ratiometric light imaging.

[0006] In addition, in order to ensure a faster switching speed of the filter wheel, usually not many filters can be designed and installed in the filter wheel. Before conducting an experiment, the filters need to be replaced in advance and preparations need to be made, which takes a lot of preparation time and the experiment cannot be switched very quickly. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a multi-applicability real-time ratiometric optical imaging device, which has good flexibility and strong applicability. With its highly integrated, multifunctional and efficient fluorescence imaging capabilities, it can perform filter switching and experimental project switching more quickly.

[0008] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0009] A multi-applicability real-time ratiometric optical imaging device, comprising:

[0010] A host, a timing controller, a light source, an image acquisition unit, a prism imaging optical path, a filter wheel, and an all-in-one illumination optical path; the host is connected to the timing controller and the image acquisition unit respectively, the timing controller receives the timing control command sent by the host, and converts the timing control command into a corresponding pulse signal, and sends a high-frequency timing signal to the image acquisition unit and the light source respectively, so as to control the on and off of the excitation light and the collection of fluorescence; the image acquisition unit is used to collect the fluorescence signal according to the timing signal, photoelectrically convert the collected fluorescence signal into a digital signal, and transmit it to the host according to the timing code; the light source is used to provide the excitation light for exciting the fluorescent drug to generate fluorescence, the all-in-one illumination optical path is used to simultaneously connect multiple light sources to the excitation illumination optical path, the filter wheel switches the corresponding filter according to the different light sources, so as to cut off the interference of the excitation light on the fluorescence imaging of the image acquisition unit; the prism imaging optical path is used to collect fluorescence, and reflect and transmit the fluorescence to the corresponding image acquisition unit according to the different spectra.

[0011] According to a multi-applicability real-time ratiometric light imaging device provided by the present invention, the prism imaging optical path includes an objective lens, a prism wheel, and a camera adapter lens. The objective lens is used to collect fluorescence, and transmit the collected fluorescence to the prism wheel after beam shaping. The prism wheel is used to switch the corresponding beam splitter prism according to the ratiometric light imaging mode, and split the fluorescence according to different spectral segments to the camera adapter lens. The camera adapter lens is used to match the camera interface and the target surface size of the imaging sensor, and converge the fluorescence transmitted from the beam splitter prism to the camera imaging sensor.

[0012] Wherein, the prism wheel comprises at least two or more beam splitter prism mounting holes, and the mounting holes are used to mount at least one beam splitter prism.

[0013] According to a multi-applicability real-time ratiometric optical imaging device provided by the present invention, the filter wheel includes at least two filter mounting holes for mounting at least one filter, which is used to cut off the interference of excitation light on camera fluorescence imaging.

[0014] According to a multi-applicability real-time ratiometric optical imaging device provided by the present invention, when a plurality of excitation light excitations and a same-band fluorescence reception method are adopted, the corresponding beam splitter prism is empty, and the fluorescence directly passes through the beam splitter prism mounting hole and is projected onto the camera adapter lens coaxial with the objective lens;

[0015] When a single excitation light is used for excitation and two different bands are received, the dichroic prism switches to the corresponding dichroic prism according to the fluorescent drug, transmits light of a certain band to the camera adapter lens coaxial with the objective lens, and reflects light of another band to the camera adapter lens perpendicular to the objective lens.

[0016] According to a multi-applicability real-time ratiometric optical imaging device provided by the present invention, when multiple excitation light excitations and fluorescence receptions in the same band are adopted, the device specifically includes:

[0017] Provide at least two light sources to access the all-in-one lighting light path;

[0018] The host sends a timing control command to the timing controller to control the switches of the two light sources. First, the first light source is controlled to emit light, and then stop emitting light. Then, the second light source is controlled to emit light, and then stop emitting light. These two processes are alternated in sequence to ensure that each light source has a chance to excite the fluorescent marker in the sample.

[0019] Select an appropriate filter by using the filter wheel, which is used to cut off the light from the two excitation light sources while allowing the fluorescence signal to pass through;

[0020] In the prism imaging optical path, the prism wheel is empty and no beam splitter prism is installed;

[0021] The timing controller synchronously controls the acquisition of the image acquisition unit; at this time, one image acquisition unit is used for acquisition, and the fluorescence signals excited by each light source are respectively acquired in a timing sequence, and these signals are converted into electrical signals and transmitted to the host;

[0022] The host receives the data from the image acquisition unit, calculates the fluorescence intensity of each pulse on each pixel, then calculates the ratio of the fluorescence intensities of adjacent pulses, and finally assigns different brightness or different pseudo colors to the calculated ratio.

[0023] According to a multi-applicability real-time ratiometric optical imaging device provided by the present invention, when a single excitation light excitation and two different waveband reception modes are adopted, the device specifically comprises:

[0024] Only one light source is used to emit light, and the light source emits light continuously or is controlled by a timing controller to emit light in a certain pulse sequence;

[0025] The two image acquisition units perform fluorescence acquisition according to a pulse sequence corresponding to the light source pulse sequence;

[0026] On the filter wheel, select and switch to the filter that cuts off the excitation light source and transmits fluorescence;

[0027] In the prism imaging optical path, the beam splitter prism at the prism wheel selects and switches to a beam splitter prism corresponding to two fluorescence receiving bands; the beam splitter prism splits the fluorescence signal received by the objective lens into two paths, one path is transmitted to the adapter lens of the first image acquisition unit, and the other path is reflected to the adapter lens of the second image acquisition unit;

[0028] The two image acquisition units receive the two-way fluorescence signals separated by the beam splitter prism through their respective adapter lenses, wherein the two image acquisition units simultaneously receive the fluorescence signals according to the wavelength range according to a certain exposure time, and convert these signals into electrical signals and transmit them to the host.

[0029] According to a multi-applicability real-time ratiometric optical imaging device provided by the present invention, before receiving data transmitted by two image acquisition units, the host needs to calibrate and align the imaging of the two image acquisition units to ensure that the images captured by the two image acquisition units can correspond one to one in the imaging field of view, that is, each pixel point can find a corresponding matching point;

[0030] After the host receives the data, the fluorescence intensity of each pixel of the two image acquisition units is calculated respectively;

[0031] According to the pixel relationship of calibration and registration, the fluorescence intensity of the corresponding pixels is proportionally calculated;

[0032] The calculated ratio is assigned different brightness or different pseudo colors, and the processed image data is transmitted to the display through the display interface for display.

[0033] According to a multi-applicability real-time ratiometric optical imaging device provided by the present invention, the ratio calculation of fluorescence intensity is expressed as the following formula:

[0034] K=Q1 mn / Q2 mn

[0035] Among them, K is the ratio, Q1 mn represents the fluorescence intensity collected by the nth pixel of the mth frame image of the camera, Q2 mn Represents the fluorescence intensity collected by the nth pixel of the mth frame image of camera 2.

[0036] According to a multi-applicability real-time ratiometric optical imaging device provided by the present invention, the light source adopts two semiconductor lasers with independent spectra, the semiconductor laser adopts an SMA905 interface as a light output interface, the semiconductor laser adopts an RS232 interface for communication, and the semiconductor laser is equipped with a BNC interface as an external trigger interface.

[0037] According to a multi-applicability real-time ratiometric optical imaging device provided by the present invention, the all-in-one illumination optical path adopts a four-in-one optical path structure, namely, four light source input channels and one light source output channel, all light source input channels and light source output channels adopt SMA905 interfaces, and in the process of light source input to light path integration, glass optical fiber is used as a transmission medium, and through optical fiber coupling technology, the light of the four light sources is efficiently coupled to a glass optical fiber for output, and the end of the output optical fiber is connected to a light outlet for outputting the integrated light to a target area, and a beam expander is used at the light outlet for light homogenization and beam expansion.

[0038] It can be seen that compared with the prior art, the device provided by the present invention has the following beneficial effects:

[0039] 1. The device of the present invention supports multiple excitation light excitations and fluorescence reception in the same band to achieve real-time ratiometric optical imaging, and also supports single excitation light excitation and reception in two different bands to achieve real-time ratiometric optical imaging, meeting different experimental requirements.

[0040] 2. When the present invention uses a single excitation light excitation and two-band reception method to achieve ratiometric light imaging, fluorescence of two bands can be received simultaneously to achieve real-time ratiometric light imaging.

[0041] 3. The present invention can switch experiments more quickly, and is particularly suitable for projects where two experiments are carried out "simultaneously", or experiments where ratiometric optical imaging analysis of multiple fluorescent substances is performed simultaneously.

[0042] 4. The device of the present invention integrates multiple key components such as a host, a timing controller, a light source, an image acquisition unit, a prism imaging optical path, a filter wheel and an all-in-one illumination optical path. The components work together to form an efficient and stable imaging system. In addition, the design of the device adopts the modular principle, so that each component can be replaced, upgraded or maintained relatively independently, which improves the flexibility and scalability of the system.

[0043] 5. The filter wheel of the present invention can switch the corresponding filter according to different light sources to cut off the interference of the excitation light; the all-in-one illumination light path allows multiple light sources to be connected at the same time, realizing the combined use of multiple excitation lights, thereby improving the applicability of the system.

[0044] 6. The present invention can obtain more accurate quantitative information by simultaneously collecting fluorescence signals of different bands and performing ratio calculation, which is of great significance for studying the dynamic changes of intracellular ions, voltage, pH, etc.

[0045] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The present invention is a schematic diagram of a filter wheel structure in the prior art.

[0047] Figure 2 It is a schematic diagram of the principle of an embodiment of a multi-applicability real-time ratiometric optical imaging device of the present invention.

[0048] Figure 3 It is a schematic diagram of the principle of the prism imaging light path in an embodiment of a multi-applicability real-time ratiometric optical imaging device of the present invention.

[0049] Figure 4 It is a schematic diagram of the principle of a prism wheel in an embodiment of a multi-applicability real-time ratiometric optical imaging device of the present invention.

[0050] Figure 5 It is an operation flow chart of an embodiment of a multi-applicability real-time ratiometric optical imaging device of the present invention.

[0051] Figure 6 It is a hardware operation schematic diagram of a first ratio light imaging mode in an embodiment of a multi-applicability real-time ratio light imaging device of the present invention.

[0052] Figure 7 It is an example principle diagram of timing commands for a first ratio light imaging mode in an embodiment of a multi-applicability real-time ratio light imaging device of the present invention.

[0053] Figure 8 It is a hardware operation schematic diagram of the second ratio light imaging mode in an embodiment of a multi-applicability real-time ratio light imaging device of the present invention.

[0054] Fig. 9 It is an example principle diagram of timing commands for the second ratio light imaging mode in an embodiment of a multi-applicability real-time ratio light imaging device of the present invention.

[0055] Fig.10 It is a schematic diagram of image calibration and registration of two cameras in an embodiment of a multi-applicability real-time ratiometric optical imaging device of the present invention.

[0056] Fig.11 It is a schematic diagram of display brightness corresponding to different ratios in an embodiment of a multi-applicability real-time ratio light imaging device of the present invention. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0059] See also Figures 2 to 11 The present invention provides a multi-applicability real-time ratiometric optical imaging device, comprising:

[0060] Host 1, timing controller 2, light source 3, image acquisition unit 4, prism imaging optical path 5, filter wheel 6, multi-in-one illumination optical path 7; host 1 is connected to timing controller 2 and image acquisition unit 4 respectively, timing controller 2 receives timing control command sent by host 1, and converts timing control command into corresponding pulse signal, and sends high-frequency timing signal to image acquisition unit 4 and light source 3 respectively, so as to control the on and off of excitation light and the collection of fluorescence; image acquisition unit 4 is used to collect fluorescence signal according to timing signal, photoelectrically convert the collected fluorescence signal into digital signal, and transmit it to host 1 according to timing coding, as shown in FIG. Figure 2 shown.

[0061] Among them, the light source 3 is used to provide excitation light for exciting fluorescent drugs to produce fluorescence, and is controlled by the timing controller 2. According to different ratio light imaging methods, one light source can emit light, or more than two light sources can emit light alternately in a timing sequence.

[0062] Among them, the multi-in-one illumination optical path 7 is used to simultaneously connect multiple light sources 3 to the excitation illumination optical path, and the filter wheel 6 switches the corresponding filter according to the different light sources 3, so as to cut off the interference of the excitation light on the fluorescence imaging of the image acquisition unit 4; the prism imaging optical path 5 is used to collect fluorescence, and reflect and transmit the fluorescence to the corresponding image acquisition unit 4 according to the different spectra; when two excitation light sources are used for excitation and one emission spectrum is received, the prism at the prism imaging optical path 5 is empty.

[0063] Among them, the host 1 sends a timing control command to the timing controller 2, and receives the image data transmitted by the image acquisition unit 4, and performs recognition, calculation, and processing of image data of different time periods and different cameras, and finally transmits the processed image data in the form of a video stream to the display 8 for display.

[0064] In this embodiment, if Figure 3As shown, the prism imaging optical path 5 includes an objective lens 9, a prism wheel 10, and a camera adapter lens 11. The objective lens 9 is used to collect fluorescence, and transmit the collected fluorescence to the prism wheel 10 after beam shaping. The prism wheel 10 is used to switch the corresponding beam splitter prism according to the ratio light imaging mode, and split the fluorescence according to different spectral segments to the camera adapter lens 11. The camera adapter lens 11 is used to match the camera interface and the target surface size of the imaging sensor, and converge the fluorescence transmitted from the beam splitter prism to the camera imaging sensor. If the camera is different, the camera adapter lens 11 may also be different;

[0065] Among them, Figure 4 As shown, the prism wheel 10 includes at least two or more dichroic prism mounting holes, which are used to install at least one dichroic prism to split fluorescence with different spectra onto two cameras respectively; the dichroic bands of the dichroic prism are related to the two bands of fluorescence received simultaneously, which are determined by the specific fluorescent drug and the test environment; the prism wheel 10 and multiple dichroic prisms are used to match the rapid switching of more than one experiment.

[0066] In this embodiment, the filter wheel 6 includes at least two or more filter mounting holes for mounting at least one filter, which is used to cut off the interference of the excitation light on the camera fluorescence imaging. For the experimental method of collecting two bands of fluorescence under the same excitation light, usually, the filter can be designed to be a bandpass or long-wave pass filter that cuts off the excitation light and allows the two fluorescence bands to pass. The filter here mainly corresponds to the filter switching between different experiments, so the switching rate requirement is not very high, and multiple mounting holes can be preset to install multiple groups.

[0067] In this embodiment, the light source 3 is usually divided into 2-4 groups according to the different emission spectra, and can be connected to the same optical path through the all-in-one illumination optical path 7 for rapid switching of the excitation light; for a higher excitation light switching rate, a semiconductor laser with external triggering or even a femtosecond laser is preferred; usually 2 groups of light sources 3 can meet the requirements of a certain experiment, and the combination of multiple groups of light sources 3 is to match the rapid switching of more than one experiment.

[0068] In this embodiment, the image acquisition units 4 are usually two groups, which may be of the same model or different models, and are used to simultaneously receive fluorescence in two different bands. In order to simplify the design of the camera adapter 11 and facilitate the alignment of the imaging between the two image acquisition units 4, cameras of the same frame model are preferred. In order to perform "real-time" dynamic observation at a higher acquisition frame rate, a scientific-grade SCMOS camera is preferred.

[0069] In this embodiment, the all-in-one illumination optical path 7 usually has 2-4 incident light interfaces, corresponding to the number of light sources 3, for simultaneously connecting multiple light sources 3; usually there are more than one output light interface, and the input incident light is emitted through these interfaces, thereby projecting onto the corresponding fluorescent drug; multiple output light interfaces are mainly considered for illumination uniformity. If there is no requirement for illumination uniformity, only one output light interface may be required.

[0070] In this embodiment, when multiple excitation lights are used for excitation and fluorescence of the same wavelength band is received, the corresponding dichroic prism is empty, and the fluorescence directly passes through the dichroic prism mounting hole and is projected onto the camera adapter lens 11 coaxial with the objective lens.

[0071] In this embodiment, when a single excitation light is used for excitation and two different bands are received, the dichroic prism switches the corresponding dichroic prism according to the fluorescent drug, transmits light of a certain band to the camera adapter lens 11 coaxial with the objective lens, and reflects light of another band to the camera adapter lens 11 perpendicular to the objective lens.

[0072] In practical applications, first, the ratio light imaging mode is selected in advance, then the fluorescent drug is selected, and then the appropriate light source 3, filter and dichroic prism are selected according to the drug, and then the light source power, camera acquisition, lens parameters and timing parameters are set. After setting, focus is performed to make the image clear, and finally normal ratio light imaging acquisition is performed, such as Figure 5 shown.

[0073] In this embodiment, when multiple excitation lights are used for excitation and fluorescence reception in the same wavelength band is used, such as Figure 6 As shown, specifically including:

[0074] Providing at least two light sources 3 connected to the all-in-one lighting light path 7;

[0075] The host 1 sends a timing control command to the timing controller 2 to control the switching of the two light sources 3, such as Figure 7 As shown, first the first light source 3 is controlled to emit light, and then stops emitting light, then the second light source 3 is controlled to emit light, and then stops emitting light, and these two processes are alternated in sequence to ensure that each light source 3 has a chance to excite the fluorescent marker in the sample;

[0076] Select an appropriate filter by using the filter wheel 6, the filter is used to cut off the light of the two excitation light sources 3, while allowing the fluorescence signal to pass through;

[0077] In the prism imaging optical path 5, the prism wheel 10 is empty, and no beam splitter prism is installed;

[0078] The timing controller 2 synchronously controls the acquisition of the image acquisition unit 4; at this time, one image acquisition unit 4 is used for acquisition, and the fluorescence signals excited by each light source 3 are respectively acquired in a timing sequence, and these signals are converted into electrical signals and transmitted to the host 1;

[0079] The host 1 receives the data from the image acquisition unit 4, calculates the fluorescence intensity of each pulse on each pixel, and then calculates the ratio of the fluorescence intensities of adjacent pulses, and finally assigns different brightness or different pseudo colors to the calculated ratio, such as Fig.11 As shown, the brightness corresponding to different ratios is schematically shown and transmitted to the display 8 through the display interface for display.

[0080] In the above method, the fluorescence corresponding to the two excitation lights is not generated at the same time, but the switching of the excitation light source is controlled by a pulse signal rather than electric filtering switching, so the light source can be switched faster than by electric filtering switching, which is closer to real-time ratiometric light imaging.

[0081] In this embodiment, when a single excitation light is used for excitation and two different wavelength bands are used for reception, Figure 8 As shown, specifically including:

[0082] This method uses only one light source 3 to emit light, and the light source 3 can emit light continuously or be controlled by a timing controller 2 to emit light according to a certain pulse sequence;

[0083] The two image acquisition units 4 perform fluorescence acquisition according to the pulse sequence corresponding to the light source pulse sequence, such as Fig. 9 As shown;

[0084] At filter wheel 6, select and switch to a filter that cuts off the excitation light source and transmits fluorescence;

[0085] In the prism imaging optical path 5, the beam splitter prism at the prism wheel 10 selects and switches to a beam splitter prism corresponding to two fluorescence receiving bands; the beam splitter prism splits the fluorescence signal received by the objective lens 9 into two paths, one of which is transmitted to the adapter lens of the first image acquisition unit 4, and the other is reflected to the adapter lens of the second image acquisition unit 4;

[0086] The two image acquisition units 4 receive the two fluorescence signals separated by the beam splitter prism through their respective adapter lenses, wherein the two image acquisition units 4 receive the fluorescence signals according to a certain exposure time and a band range at the same time (the data collected each time exposure corresponds to a frame of image), and convert these signals into electrical signals and transmit them to the host 1.

[0087] According to a multi-applicability real-time ratiometric optical imaging device provided by the present invention, before receiving the data transmitted by the two image acquisition units 4, the host 1 needs to calibrate and align the images of the two image acquisition units 4, such as Fig.10 As shown, it is ensured that the images captured by the two image acquisition units 4 can correspond one to one in the imaging field of view, that is, each pixel point can find a corresponding matching point;

[0088] After receiving the data, the host 1 calculates the fluorescence intensity of each pixel of the two image acquisition units 4 respectively;

[0089] According to the pixel relationship of calibration and registration, the fluorescence intensity of the corresponding pixels is proportionally calculated (fluorescence intensity collected by camera one / fluorescence intensity collected by camera two);

[0090] The calculated ratio is assigned different brightness or different pseudo colors, and the processed image data is transmitted to the display 8 through the display interface for display.

[0091] In this example, the ratio of fluorescence intensity is calculated as follows:

[0092] K=Q1 mn / Q2 mn

[0093] Among them, K is the ratio, Q1 mn represents the fluorescence intensity collected by the nth pixel of the mth frame image of the camera, Q2 mn Represents the fluorescence intensity collected by the nth pixel of the mth frame image of camera 2.

[0094] In the above method, fluorescence in two bands is generated and received simultaneously, so it is a true real-time ratiometric optical imaging method.

[0095] In this embodiment, after the ratiometric light imaging experiment test of a certain drug is completed, it is necessary to perform an imaging test on another drug, which usually requires switching the light source, the filter, and the dichroic prism.

[0096] Because the light source 3 is integrated through the all-in-one illumination optical path 7, it can be quickly switched by directly selecting the host parameters, the filter can be quickly switched by directly controlling the filter wheel 6 through the host parameter selection, and the dichroic prism can be quickly switched by directly controlling the prism wheel 10 through the host parameter selection, thereby ensuring that the device of the present invention can quickly switch between different fluorescent drug experiments.

[0097] Furthermore, the host 1 is a computer with a Windows system and a discrete graphics card, which receives and communicates image data through a USB interface, performs data calculation and image display output through a GPU, and performs logic control through a CPU.

[0098] Furthermore, the timing controller uses a conventional signal generator with an output frequency ≥10MHz, performs external trigger control through a 4-channel BNC interface, 2 channels are connected to the light source, 2 channels are connected to the camera, and receives host communication through a USB interface.

[0099] Furthermore, the light source 3 uses two semiconductor lasers with independent spectra, the semiconductor laser uses an SMA905 interface as a light output interface, the semiconductor laser uses an RS232 interface for communication, and the semiconductor laser is equipped with a BNC interface as an external trigger interface.

[0100] Furthermore, the all-in-one lighting optical path 7 adopts a four-in-one optical path structure, namely, four light source input channels and one light source output channel. All light source input channels and light source output channels adopt SMA905 interfaces. In the process of inputting the light source 3 into the optical path integration, glass optical fiber is used as the transmission medium. Through the optical fiber coupling technology, the light from the four light sources is efficiently coupled into a glass optical fiber for output. The end of the output optical fiber is connected to a light outlet for outputting the integrated light to the target area. A beam expander is used at the light outlet for light homogenization and beam expansion.

[0101] Furthermore, the image acquisition unit 4 uses two SCMOS cameras of the same model, with a USB interface for data transmission and communication and a BNC interface for external trigger control.

[0102] Furthermore, the filter wheel 6 is installed at the front end of the objective lens, driven by a stepper motor and belt transmission, and communicated by an RS232 interface. The filter wheel 6 has 6 holes, and a filter with a diameter of 50 can be installed to avoid blocking the field of view and affecting the reception of fluorescence; the filter is customized or selected according to the fluorescent drug, and preferably the filter with a transmittance greater than 90% and a cutoff greater than OD4;

[0103] Furthermore, the prism imaging optical path 5 and the objective lens are customized or selected according to the optical path, the camera adapter lens 11 is customized, the prism wheel 10 is driven by a stepper motor and a belt drive, and an RS232 interface is used for communication. There are 4 holes for the dichroic prism, and the size matches the objective lens 9 and the camera adapter lens 11. The dichroic prism is customized to meet the corresponding size and dichroic spectrum requirements.

[0104] In addition, the host 1, the timing controller 2, and the excitation light source 3 can be three independent devices that communicate through an external interface, or they can be integrated devices that communicate through an internal interface;

[0105] The light source 3 can be a laser light source or a monochromatic LED light source, etc., whichever meets the requirements of the excitation spectrum range, excitation light intensity and light emission timing control.

[0106] The host 1 may be based on an operating system such as Windows or Linux, and the data processing and calculation may be based on a CPU, a GPU or an FPGA.

[0107] On the basis of the above, the filter wheel 6 can also be installed between the camera and the camera adapter lens 11.

[0108] In summary, the device of the present invention supports real-time ratiometric optical imaging by means of multiple excitation light excitations and fluorescence reception in the same band, and also supports real-time ratiometric optical imaging by means of single excitation light excitation and reception in two different bands, thus meeting different experimental requirements.

[0109] Furthermore, when the present invention uses a single excitation light excitation and two-band reception method to achieve ratiometric optical imaging, fluorescence of two bands can be received simultaneously to achieve real-time ratiometric optical imaging.

[0110] Furthermore, the present invention can switch experiments more quickly, and is particularly suitable for projects where two experiments are carried out "simultaneously", or experiments where ratiometric optical imaging analysis of multiple fluorescent substances is performed simultaneously.

[0111] Furthermore, the device of the present invention integrates multiple key components such as a host 1, a timing controller 2, a light source 3, an image acquisition unit 4, a prism imaging optical path 5, a filter wheel 6 and an all-in-one illumination optical path 7, and the components work together to form an efficient and stable imaging system. In addition, the design of the device adopts a modular principle, so that each component can be replaced, upgraded or maintained relatively independently, thereby improving the flexibility and scalability of the system.

[0112] Furthermore, the filter wheel 6 of the present invention can switch the corresponding filter according to the different light sources 3 to cut off the interference of the excitation light; the all-in-one illumination light path 7 allows multiple light sources 3 to be connected at the same time, realizing the combined use of multiple excitation lights, thereby improving the applicability of the system.

[0113] Furthermore, the present invention can obtain more accurate quantitative information by simultaneously collecting fluorescence signals of different bands and performing ratio calculations, which is of great significance for studying the dynamic changes of intracellular ions, voltage, pH, etc.

[0114] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0115] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A multi-applicability real-time ratiometric optical imaging device, characterized in that: include: Host, timing controller, light source, image acquisition unit, prism imaging optical path, filter wheel, all-in-one illumination optical path; The host is connected to the timing controller and the image acquisition unit respectively. The timing controller receives the timing control command sent by the host, converts the timing control command into a corresponding pulse signal, and sends a high-frequency timing signal to the image acquisition unit and the light source respectively to control the on and off of the excitation light and the collection of fluorescence; the image acquisition unit is used to collect the fluorescence signal according to the timing signal, photoelectrically convert the collected fluorescence signal into a digital signal, and transmit it to the host according to the timing code; The light source is used to provide excitation light for exciting fluorescent drugs to produce fluorescence, the all-in-one illumination light path is used to simultaneously connect multiple light sources to the excitation illumination light path, and the filter wheel switches the corresponding filter according to the different light sources to cut off the interference of the excitation light on the fluorescence imaging of the image acquisition unit; the prism imaging light path is used to collect fluorescence, and reflect and transmit the fluorescence to the corresponding image acquisition unit according to the different spectra.

2. The device according to claim 1, characterized in that: The prism imaging optical path includes an objective lens, a prism wheel, and a camera adapter lens. The objective lens is used to collect fluorescence and transmit the collected fluorescence to the prism wheel after beam shaping. The prism wheel is used to switch the corresponding beam splitter prism according to the ratio light imaging mode, and split the fluorescence according to different spectral segments to the camera adapter lens. The camera adapter lens is used to match the camera interface and the target surface size of the imaging sensor, and converge the fluorescence transmitted from the beam splitter prism to the camera imaging sensor. Wherein, the prism wheel comprises at least two or more beam splitter prism mounting holes, and the mounting holes are used to mount at least one beam splitter prism.

3. The device according to claim 1, characterized in that: The filter wheel includes at least two filter installation holes for installing at least one filter, and the filter is used to cut off the interference of the excitation light on the camera fluorescence imaging.

4. The device according to claim 2, characterized in that: When multiple excitation lights are used for excitation and fluorescence reception in the same wavelength band, the corresponding beam splitter prism is empty, and the fluorescence directly passes through the beam splitter prism mounting hole and is projected onto the camera adapter lens coaxial with the objective lens; When a single excitation light is used for excitation and two different bands are received, the dichroic prism switches to the corresponding dichroic prism according to the fluorescent drug, transmits light of a certain band to the camera adapter lens coaxial with the objective lens, and reflects light of another band to the camera adapter lens perpendicular to the objective lens.

5. The device according to claim 4, characterized in that: When multiple excitation lights are used for excitation and fluorescence reception in the same wavelength band, the specific methods include: Provide at least two light sources to access the all-in-one lighting light path; The host sends a timing control command to the timing controller to control the switches of the two light sources. First, the first light source is controlled to emit light, and then stop emitting light. Then, the second light source is controlled to emit light, and then stop emitting light. These two processes are alternated in sequence to ensure that each light source has a chance to excite the fluorescent marker in the sample. Select an appropriate filter by using the filter wheel, which is used to cut off the light from the two excitation light sources while allowing the fluorescence signal to pass through; In the prism imaging optical path, the prism wheel is empty and no beam splitter prism is installed; The timing controller synchronously controls the acquisition of the image acquisition unit; at this time, one image acquisition unit is used for acquisition, and the fluorescence signals excited by each light source are respectively acquired in a timing sequence, and these signals are converted into electrical signals and transmitted to the host; The host receives the data from the image acquisition unit, calculates the fluorescence intensity of each pulse on each pixel, then calculates the ratio of the fluorescence intensities of adjacent pulses, and finally assigns different brightness or different pseudo colors to the calculated ratio.

6. The device according to claim 4, characterized in that: When a single excitation light is used for excitation and two different wavelength bands are used for reception, the specific steps include: Only one light source is used to emit light, and the light source emits light continuously or is controlled by a timing controller to emit light in a certain pulse sequence; The two image acquisition units perform fluorescence acquisition according to a pulse sequence corresponding to the light source pulse sequence; On the filter wheel, select and switch to the filter that cuts off the excitation light source and transmits fluorescence; In the prism imaging optical path, the beam splitter prism at the prism wheel selects and switches to a beam splitter prism corresponding to two fluorescence receiving bands; the beam splitter prism splits the fluorescence signal received by the objective lens into two paths, one path is transmitted to the adapter lens of the first image acquisition unit, and the other path is reflected to the adapter lens of the second image acquisition unit; The two image acquisition units receive the two-way fluorescence signals separated by the beam splitter prism through their respective adapter lenses, wherein the two image acquisition units simultaneously receive the fluorescence signals according to the wavelength range according to a certain exposure time, and convert these signals into electrical signals and transmit them to the host.

7. The device according to claim 6, characterized in that: Before receiving the data from the two image acquisition units, the host needs to calibrate and align the images of the two image acquisition units to ensure that the images taken by the two image acquisition units can correspond one to one in the imaging field of view, that is, each pixel point can find the corresponding matching point; After the host receives the data, the fluorescence intensity of each pixel of the two image acquisition units is calculated respectively; According to the pixel relationship of calibration and registration, the fluorescence intensity of the corresponding pixels is proportionally calculated; The calculated ratio is assigned different brightness or different pseudo colors, and the processed image data is transmitted to the display through the display interface for display.

8. The device according to claim 5 or 7, characterized in that: The ratio of fluorescence intensity is calculated as follows: K=Q1 mn / Q2 mn Among them, K is the ratio, Q1 mn represents the fluorescence intensity collected by the nth pixel of the mth frame image of the camera, Q2 mn Represents the fluorescence intensity collected by the nth pixel of the mth frame image of camera 2.

9. The device according to any one of claims 1 to 7, characterized in that: The light source adopts semiconductor lasers with two independent spectra, the semiconductor laser adopts SMA905 interface as light output interface, the semiconductor laser adopts RS232 interface for communication, and the semiconductor laser is equipped with BNC interface as external trigger interface.

10. The device according to any one of claims 1 to 7, characterized in that: The all-in-one illumination optical path adopts a four-in-one optical path structure, namely, four light source input channels and one light source output channel. All light source input channels and light source output channels adopt SMA905 interfaces. In the process of light source input to the optical path integration, glass optical fiber is used as a transmission medium. Through the optical fiber coupling technology, the light of the four light sources is efficiently coupled to a glass optical fiber for output. The end of the output optical fiber is connected to a light outlet for outputting the integrated light to the target area. A beam expander is used at the light outlet for light homogenization and beam expansion.

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