Neuron activity high-speed fluorescence imaging system based on event camera microscope

Abstract

The invention relates to a neuron activity high-speed fluorescence imaging system based on an event camera microscope, which comprises a light source, a fluorescence excitation module and a fluorescence imaging module which are arranged, and an event camera is adopted in the fluorescence imaging module for image acquisition; the light source illuminates a sample through the fluorescence excitation module, and fluorescence on the sample emits a signal and then is imaged on the event camera through the fluorescence imaging module. Compared with the prior art, the high-speed recording of the neuron dynamic information of the biological tissue sample can be realized on the premise of not sacrificing the imaging view, and the device has the advantages of low background fluorescence and simple and easy operation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescence microscopic imaging, in particular to a neuron activity high-speed fluorescence imaging system based on an event camera microscope. BACKGROUND

[0002] Fluorescence microscopic imaging technology includes wide-field fluorescence microscope, confocal microscope, multi-photon microscope, light field microscope, light sheet microscope, etc. The basic principle is that the light of a specific wavelength irradiates the fluorescent group, which will emit fluorescence. The emitted fluorescence can be collected by a photon detector to realize the imaging of fluorescence and obtain the structure information or functional information of the biological tissue labeled by the fluorescent group.

[0003] The advantage of applying fluorescence microscope to the structure or functional imaging of biological tissue is that it has high resolution and can perform non-invasive imaging on tissue samples. At present, fluorescence microscope has been widely used in neuron calcium activity detection and recording experiments. However, the response of Ca 2+ to the action potential of neurons is indirect rather than direct; the activity of Ca 2+ lags behind the action potential; and the response time of Ca 2+ is relatively long (about hundreds of milliseconds to 1 second), while the response time of neuron electrical signal is within a few milliseconds, so recording the calcium activity information in neurons cannot accurately reflect the real activity information of neurons. At present, a large number of voltage fluorescence probes based on ion channels have been invented, and the fluorescence response speed can match the change of membrane potential, but due to the imaging limitation of fluorescence microscope, there are still problems such as poor imaging effect, slow imaging speed or complex equipment difficult to be commercialized.

[0004] After decades of continuous development, a variety of high-speed fluorescence imaging methods have been invented and applied to the monitoring and recording of neuron calcium activity and voltage activity. However, due to the certain shortcomings of various methods, such as small imaging field, complex device, high cost, etc., they have not been widely applied in the field of neuroscience.

[0005] At present, the optical detection means of voltage fluorescence probes commonly used by researchers in the field of neurobiology still mainly relies on the relatively traditional, simple and easy-to-operate, and easy-to-build wide-field fluorescence microscope. The imaging speed mainly depends on the inherent sampling frame rate of the camera. The specific method is to improve the sampling rate by sacrificing the imaging field. And due to the problems of poor resolution and strong background fluorescence signal of wide-field microscope, it is only suitable for samples with sparse fluorescent markers.

[0006] Therefore, there is an urgent need for a fluorescence microscopic imaging device that can realize high-speed fluorescence signal acquisition, low background fluorescence, and simple and easy-to-operate equipment. SUMMARY

[0007] The purpose of the present invention is to overcome the defects of the above-mentioned existing technologies and provide a high-speed fluorescence imaging system for neuronal activity based on an event camera microscope, which can achieve high-speed recording of dynamic information of neurons in biological tissue samples without sacrificing the imaging field of view, and has the advantages of low background fluorescence and simple and easy operation of the equipment.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The present invention provides a high-speed fluorescence imaging system for neuronal activity based on an event camera microscope, comprising a light source, a fluorescence excitation module and a fluorescence imaging module. The fluorescence imaging module uses an event camera for image acquisition; the light source illuminates the sample through the fluorescence excitation module, and the fluorescence emission signal on the sample is then imaged onto the event camera through the fluorescence imaging module.

[0010] Preferably, the light source includes an LED light source, a laser light source or a mercury lamp.

[0011] Preferably, the fluorescence excitation module includes a lens, a filter, a dichroic mirror and a microscope objective lens arranged in sequence; the light beam emitted by the light source is expanded to a set spot size through the lens, and the expanded light beam is reflected onto the microscope objective lens after passing through the filter and the dichroic mirror, and illuminates the sample through the microscope objective lens to form a fluorescence excitation light path.

[0012] Preferably, the system is further provided with a three-dimensional translation stage for adjusting the sample to the focal plane of the microscope objective lens, and adjusting the imaging region of interest to be within the field of view by adjusting the three-dimensional translation stage.

[0013] Preferably, an optical element for adjusting the size and direction of the light spot in the light path is further provided between the lens and the light source.

[0014] Preferably, at least one set of filter-tube lens-event camera combination is provided in the fluorescence imaging module.

[0015] Preferably, in the filter-tube lens-event camera combination, the relative positions of the filter and the tube lens are not unique.

[0016] Preferably, the fluorescence imaging module is provided with at least one set of filter-tube lens-event camera combination and at least one set of filter-tube lens-ordinary camera combination, which are respectively used to simultaneously receive neuronal fluorescence activity signals and full-frame fluorescence signals.

[0017] Preferably, when a filter-tube lens-event camera combination and a filter-tube lens-ordinary camera combination are provided in the fluorescence imaging module, a spectroscopic element is used to distribute the fluorescence reflection signal in proportion.

[0018] Preferably, the light splitting element comprises a beam splitter or a dichroic mirror.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] 1) The event camera is used to record dynamic information of the fluorescent signal, and has higher imaging speed than a general camera, thereby realizing high-time-resolution imaging of neuron activity.

[0021] 2) Based on the inherent imaging principle and mechanism of the event camera, the event camera has no imaging and collecting ability for information without light intensity change, and during the imaging of the biological tissue, the spontaneous background fluorescence generated by the biological tissue will not be recorded by the event camera, so that the present application can reduce the background fluorescence signal and realize a high signal-to-background ratio.

[0022] 3) The general camera and the event camera are introduced at the same time to simultaneously receive the neuron fluorescent activity signal and the full-width fluorescent signal, and the light splitting element is used to proportionally distribute the fluorescent signal to the two cameras at the same time, so as to realize convenient sample searching and positioning of the region of interest.

[0023] 4) By setting multiple groups of filter-tube lens-event camera combinations, different color fluorescent signals can be received at the same time, and more fluorescent signals can be simultaneously imaged.

[0024] 5) The three-dimensional displacement table is equipped for adjusting the position of the sample, adjusting the sample to the focal plane of the microscope objective, and adjusting the imaging region of interest to the field of view by adjusting the three-dimensional displacement table, so as to realize convenient sample searching and positioning of the region of interest.

[0025] 6) The present application can also be applied to time-focused two-photon wide-field imaging, which can remove the blurring effect of the fluorescent signal generated by the non-focal plane on imaging, so that when the neuron activity recording probe is used, the problem of reducing fluorescent crosstalk by diluting the fluorescent probe is not needed.

[0026] 7) The fluorescent imaging system proposed in the present application has the advantages of simple structure, easy to build, low cost, strong stability and good universality. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a structure schematic diagram of the neuron activity high-speed fluorescent imaging system in embodiment 1;

[0028] Figure 2 It is a structure schematic diagram of the neuron activity high-speed fluorescent imaging system in embodiment 2;

[0029] Figure 3 It is a structure schematic diagram of the neuron activity high-speed fluorescent imaging system in embodiment 3;

[0030] Figure 4 Structure diagram of neuron activity high-speed fluorescence imaging system in embodiment 4;

[0031] Reference signs:

[0032] 101-light source, 102-first lens, 103-second lens, 104-first filter, 105-dichroic mirror, 106-microscope objective, 107-second filter, 108-barrel lens, 109-event camera;

[0033] 201-light source, 202-first lens, 203-second lens, 204-first filter, 205-first dichroic mirror, 206-microscope objective, 207-second dichroic mirror, 208-second filter, 209-first barrel lens, 210-first event camera, 211-third filter, 212-second barrel lens, 213-second event camera;

[0034] 301-light source, 302-first lens, 303-second lens, 304-first filter, 305-dichroic mirror, 306-microscope objective, 307-beam splitter, 308-second filter, 309-first barrel lens, 310-first event camera, 311-third filter, 312-second barrel lens, 313-ordinary camera;

[0035] 401-light source, 402-first light splitting element, 403-second light splitting element, 404-first lens, 405-second lens, 406-dichroic mirror, 407-microscope objective, 408-second dichroic mirror, 409-first filter, 410-first barrel lens, 411-event camera, 412-second filter, 413-second barrel lens, 414-second event camera. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should fall within the scope of protection of the present application.

[0037] Embodiment 1

[0038] The embodiment provides a neuron activity high-speed fluorescence imaging system based on an event camera microscope, which comprises a light source, a fluorescence excitation module and a fluorescence imaging module. The event camera is used for image acquisition in the fluorescence imaging module. The light source illuminates on a sample through the fluorescence excitation module, and the fluorescence emission signal on the sample is imaged to the event camera through the fluorescence imaging module.

[0039] The fluorescence excitation module comprises, in sequence, a lens, a filter, a dichroic mirror and a microscope objective; the light beam emitted by the light source is expanded to a set spot size by the lens, the expanded light beam is reflected onto the microscope objective after passing through the filter and the dichroic mirror, and is illuminated on the sample after passing through the microscope objective, thereby forming a fluorescence excitation light path.

[0040] In this embodiment, a set of filter-barrel lens-event camera combination is arranged in the fluorescence imaging module, as shown in the figure. Figure 1 The neuron activity high-speed fluorescence imaging system based on the event camera microscope of this embodiment specifically comprises: a light source 101, a first lens 102, a second lens 103, a first filter 104, a dichroic mirror 105, a microscope objective 106, a second filter 107, a barrel lens 108 and an event camera 109.

[0041] The light path of the fluorescence excitation module is arranged as follows: the excitation light beam emitted by the light source 101 is expanded to an appropriate spot size after passing through the first lens 102 and the second lens 103, the expanded light beam is reflected onto the microscope objective 106 after passing through the first filter 104 and the dichroic mirror 105, and finally, is illuminated on the sample after passing through the microscope objective 106.

[0042] The light path of the fluorescence imaging module is arranged as follows: after the fluorescence on the sample is irradiated by the excitation light beam, the emitted fluorescence is reflected back to the microscope objective 106, and after being collected by the microscope objective 106, it passes through the dichroic mirror 105, the second filter 107 and the barrel lens 108 in sequence, and finally is imaged onto the photosensitive unit of the event camera 109.

[0043] The light source 101 in this embodiment can use a laser light source, an LED light source or a mercury lamp combined with a filter combination.

[0044] The relative positions of the second filter 107 and the barrel lens 108 of this embodiment can be interchanged.

[0045] This embodiment is also equipped with a three-dimensional displacement stage for adjusting the position of the sample, adjusting the sample to the focal plane of the microscope objective 106, and adjusting the imaging area of interest to the field of view range by adjusting the three-dimensional displacement stage.

[0046] This embodiment utilizes the high-speed imaging performance of the event camera, and the designed neuron activity high-speed fluorescence imaging system can realize high-speed imaging of the fluorescence signal with neuron activity; and since the event camera does not have the ability to image the background fluorescence signal whose brightness does not change with time, the acquired image almost records no spontaneous fluorescence generated by the sample, greatly reducing the background signal generated by the spontaneous fluorescence, thereby realizing an image with high signal-to-background ratio.

[0047] In addition, the embodiment can also be applied to time-focused two-photon wide-field imaging, which can remove the blurring effect of fluorescent signals generated by non-focal planes on imaging, thereby eliminating the problem of reducing fluorescent crosstalk by diluting fluorescent probes when using neuron activity recording probes.

[0048] Embodiment 2

[0049] The difference between the present embodiment and Embodiment 1 is that a plurality of filter-tube lens-event camera combinations are arranged in the fluorescent imaging module in the present embodiment.

[0050] As shown in Figure 2 The present embodiment takes two filter-tube lens-event camera combinations as an example to illustrate a neuron activity high-speed fluorescent imaging system based on an event camera microscope in detail. The system includes a light source 201, a first lens 202, a second lens 203, a first filter 204, a first dichroic mirror 205, a microscope objective 206, a second dichroic mirror 207, a second filter 208, a first tube lens 209, a first event camera 210, a third filter 211, a second tube lens 212, and a second event camera 213.

[0051] The optical path of the fluorescent excitation module: the excitation light beam emitted by the light source 201 is expanded to an appropriate spot size after passing through the first lens 202 and the second lens 203, and the expanded light beam is reflected onto the microscope objective 206 after passing through the first filter 204 and the first dichroic mirror 205. Finally, the light is illuminated onto the sample after passing through the microscope objective 206.

[0052] The optical path of the fluorescent imaging module: the fluorescent light emitted after the sample is illuminated by the excitation light beam is reflected back to the microscope objective 206, and after being collected by the microscope objective 206, it passes through the first dichroic mirror 205. The fluorescent light in a specific wavelength range is transmitted through the second dichroic mirror 207, and then sequentially passes through the second filter 208 and the first tube lens 209, and finally imaged onto the photosensitive unit of the first event camera 210.

[0053] The fluorescent light of other wavelengths is reflected by the second dichroic mirror 207, sequentially passes through the third filter 211 and the second tube lens 212, and finally imaged onto the photosensitive unit of the first event camera 213.

[0054] The relative positions between the first filter 208 and the first tube lens 209, and the third filter 211 and the second tube lens 212 in the present embodiment can be interchanged.

[0055] The optical films coated on the first dichroic mirror 205 and the second dichroic mirror 207 in the present embodiment have different applicable wavelengths.

[0056] In this embodiment, the wavelength ranges used by the second filter 208 and the third filter 211 are also different, and their selection is adapted based on the reflection band and the transmission band of the second dichroic mirror 207 .

[0057] In this embodiment, two sets of filter-tube lens-event camera combinations are provided for simultaneously receiving fluorescence signals of different colors. Simultaneous imaging of more fluorescence signals can be achieved by further adding filter-tube lens-event camera combinations and adding dichroic mirrors.

[0058] The other configurations of this embodiment are the same as those of embodiment 1.

[0059] Implementation Case 3

[0060] The difference between this embodiment and embodiment 2 is that at least one set of filter-tube lens-event camera combination and at least one set of filter-tube lens-ordinary camera combination are simultaneously provided in the fluorescence imaging module.

[0061] like Figure 3 As shown, taking a set of filter-tube lens-event camera combination and a set of filter-tube lens-ordinary camera combination as examples, a high-speed fluorescence imaging system of neuronal activity based on event camera microscope is described in detail. The system includes: a light source 301, a first lens 302, a second lens 303, a first filter 304, a dichroic mirror 305, a microscope objective 306, a beam splitter 307, a second filter 308, a first tube lens 309, a first event camera 310, a third filter 311, a second tube lens 312 and an ordinary camera 313.

[0062] The optical path of the fluorescence excitation module is as follows: the excitation light beam emitted by the light source 301 passes through the first lens 302 and the second lens 303 and is expanded to an appropriate spot size. The expanded light beam passes through the first filter 304 and the dichroic mirror 305 and is reflected onto the microscope objective lens 306. Finally, it passes through the microscope objective lens 306 and illuminates the sample.

[0063] The optical path of the fluorescence imaging module is as follows: after the fluorescence on the sample is irradiated by the excitation light beam, the emitted fluorescence is reflected back to the microscope objective 306, collected by the microscope objective 306, and passes through the dichroic mirror 305. After that, a portion of the fluorescence is transmitted through the beam splitter 307, passes through the second filter 308 and the first tube lens 309 in sequence, and is finally imaged onto the photosensitive unit of the event camera 310.

[0064] Another part of the fluorescence is reflected by the beam splitter 307 , passes through the third filter 311 and the second tube lens 312 in sequence, and is finally imaged onto the photosensitive unit of the camera 313 .

[0065] In this embodiment, the ordinary camera 313 can be a CCD camera, an sCMOS camera, an EMCCD camera, etc.

[0066] In this embodiment, the beam splitter 307 can be freely selected to have different transmission and reflection ratios.

[0067] The filter-tube lens-event camera combination and the filter-tube lens-camera combination provided in this embodiment are used to simultaneously receive neuronal fluorescence activity signals and full-frame fluorescence signals, respectively. By continuing to add filter-tube lens-event camera / camera combinations and adding dichroic mirrors or beam splitters, simultaneous imaging of more color fluorescence activities and full-frame fluorescence signals can be achieved.

[0068] The other configurations of this embodiment are the same as those of embodiment 2.

[0069] Implementation Case 4

[0070] like Figure 4 As shown, the high-speed fluorescence imaging system of neuronal activity based on event camera microscope provided in this embodiment includes a light source 401, a first spectroscopic element 402, a second spectroscopic element 403, a first lens 404, a second lens 405, a first dichroic mirror 406, a microscope objective 407, a second dichroic mirror 408, a first filter 409, a first tube lens 410, a first event camera 411, a second filter 412, a second tube lens 413 and a second event camera 414.

[0071] The optical path of the fluorescence excitation module is as follows: the excitation light beam emitted by the light source 401 passes through the first spectrometer 402 and the second spectrometer 403, and then is widened. The widened excitation light then passes through the first lens 404 and the second lens 405 in sequence and is expanded to an appropriate spot size. The expanded light beam passes through the first dichroic mirror 406 and is reflected onto the microscope objective lens 407. Finally, it passes through the microscope objective lens 407 and illuminates the sample.

[0072] The optical path of the fluorescence imaging module is as follows: after the fluorescence on the sample is irradiated by the excitation light beam, the emitted fluorescence is reflected back to the microscope objective lens 407, collected by the microscope objective lens 407, and passes through the first dichroic mirror 406. After that, the fluorescence within a specific wavelength range is transmitted through the second dichroic mirror 408, passes through the first filter 409 and the first tube lens 410 in sequence, and is finally imaged onto the photosensitive unit of the first event camera 411.

[0073] Fluorescence of other wavelengths is reflected by the second dichroic mirror 408 , passes through the second filter 412 and the second tube lens 413 in sequence, and is finally imaged onto the photosensitive unit of the second event camera 414 .

[0074] In this embodiment, the optical films coated on the first dichroic mirror 406 and the second dichroic mirror 408 have different applicable wavelengths.

[0075] The wavelength ranges used by the first filter 409 and the second filter 412 in this embodiment are also different, and are selected according to the reflection band and the transmission band of the second dichroic mirror 408.

[0076] The second dichroic mirror 408 in this embodiment can be replaced by a beam splitter according to requirements. The second event camera 414 in this embodiment can be replaced by a normal camera (such as a CCD camera, an sCMOS camera, an EMCCD camera, etc.) according to requirements.

[0077] The other settings of this embodiment are the same as those of Embodiment 2.

[0078] The above merely provides a specific implementation of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of various equivalent modifications or replacements within the technical range disclosed by the present application, and these modifications or replacements should be encompassed within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A neuron activity high-speed fluorescence imaging system based on an event camera microscope, characterized in that, The system comprises a light source, a fluorescence excitation module and a fluorescence imaging module, the fluorescence imaging module uses an event camera to collect images; the light source illuminates the sample through the fluorescence excitation module, and the fluorescence emission signal on the sample is imaged to the event camera through the fluorescence imaging module.

2. The neuron activity high-speed fluorescence imaging system based on an event camera microscope according to claim 1, wherein, The light source comprises an LED light source, a laser light source or a mercury lamp.

3. The neuron activity high-speed fluorescence imaging system based on an event camera microscope according to claim 1, wherein, The fluorescence excitation module comprises a lens, a filter, a dichroic mirror and a microscope objective arranged in sequence; the light beam emitted by the light source is expanded to a set spot size through the lens, the expanded light beam is reflected to the microscope objective through the filter and the dichroic mirror, and the sample is illuminated through the microscope objective to form a fluorescence excitation light path.

4. The neuron activity high-speed fluorescence imaging system based on an event camera microscope according to claim 3, characterized in that, The system is further provided with a three-dimensional displacement table for adjusting the sample to the focal plane of the microscope objective and adjusting the imaging area of interest to the field of view by adjusting the three-dimensional displacement table.

5. The neuron activity high-speed fluorescence imaging system based on an event camera microscope according to claim 1, wherein, An optical element for adjusting the spot size and direction in the optical path is further arranged between the lens and the light source.

6. The neuron activity high-speed fluorescence imaging system based on an event camera microscope according to claim 1, wherein, At least one filter-barrel lens-event camera combination is arranged in the fluorescence imaging module.

7. The neuron activity high-speed fluorescence imaging system based on an event camera microscope according to claim 6, wherein, The relative positions of the filter and the barrel lens in the filter-barrel lens-event camera combination are not unique.

8. The neuron activity high-speed fluorescence imaging system based on an event camera microscope according to claim 6, wherein, At least one filter-barrel lens-event camera combination and at least one filter-barrel lens-ordinary camera combination are arranged in the fluorescence imaging module at the same time, and are respectively used to receive the neuronal fluorescence activity signal and the full-frame fluorescence signal at the same time.

9. The neuron activity high-speed fluorescence imaging system based on an event camera microscope according to claim 8, wherein, When the filter-barrel lens-event camera combination and the filter-barrel lens-ordinary camera combination are arranged in the fluorescence imaging module at the same time, a light splitting element is used to distribute the fluorescence reflection signal in proportion.

10. The neuron activity high-speed fluorescence imaging system based on an event camera microscope according to claim 9, wherein, The light splitting element comprises a beam splitter or a dichroic mirror.

Images