CN112075925A

CN112075925A - Fluorescent imaging illumination device, imaging system and imaging method based on speckle principle

Info

Publication number: CN112075925A
Application number: CN202010993369.2A
Authority: CN
Inventors: 郭青春; 张垒; 王姝
Original assignee: Beijing Brain Science And Brain Like Research Center
Current assignee: Beijing Brain Science And Brain Like Research Center
Priority date: 2020-09-21
Filing date: 2020-09-21
Publication date: 2020-12-15

Abstract

The invention discloses a fluorescent imaging illumination device, an imaging system and an imaging method based on a speckle principle. The fluorescence imaging illumination device comprises a coupling fiber bundle and one or more mixed excitation light generation units. The imaging system comprises the fluorescent imaging illumination device based on the speckle principle, a mirror body, an imaging device and an acquisition control device. The imaging method comprises the following steps: and controlling a fluorescence imaging illumination device of the system to alternately generate fluorescence excitation light for forming speckle illumination and fluorescence excitation light for forming uniform light illumination, controlling the imaging device to perform speckle illumination imaging and uniform light illumination imaging, and obtaining an optical tomography by adopting a HiLo optical tomography algorithm. On the basis of the existing wearable fluorescence microscope system, the invention adopts a method of mixing coherent light source and incoherent light source for illumination to obtain the optical tomography image of the target sample, thereby improving the contrast and resolution of the imaging and expanding the application range of the wide-field fluorescence microscope system.

Description

Fluorescent imaging illumination device, imaging system and imaging method based on speckle principle

Technical Field

The invention belongs to the field of biological imaging, and particularly relates to a fluorescent imaging illumination device, an imaging system and an imaging method based on a speckle principle.

Background

Imaging brain cells in conscious and freely moving animals is an important tool in the field of neuroscience for studying the link between animal behavior and cerebral neurons. During the corresponding behavior process of the animal, the action potential change of the neurons in the specific area of the brain area of the animal is observed, so that the relation between the animal behavior and the information processing of the neurons, the loops and the higher-level neurons can be researched. Therefore, the detection of the neuron activity signals is the basis of neuroscience research, and conventional means such as electrophysiological recording have the problems of high technical difficulty, low flux, no specificity, lack of spatial resolution and the like. The development of optical nerve probes is an important supplement to modern neuroscience research and detection technologies, optical signals can provide high spatial and temporal resolution, are less invasive than traditional microelectrode methods, and can be used for in vivo research on the functions of neurons in more natural behaviors.

By matching with corresponding optical fluorescent probes, various fluorescence microscopic techniques can be adopted to perform monochromatic or multicolor neuron activity imaging, and in-situ optogenetic stimulation can be performed simultaneously by increasing optogenetic stimulation light sources. In order to record the nerve function of animals in natural behaviors under the conditions of wakefulness, no restriction and mobility, a miniaturized fluorescence microscope implemented by utilizing a micro optical device, a micro imaging element and a micro mirror body structure has been developed in the industry, the micro fluorescence microscope has small volume, the mirror body can be fixed on the head of a mouse, the weight of the micro fluorescence microscope does not influence the free activity of the mouse, and the micro fluorescence microscope is applied to the research of the nerve science at home and abroad at present. The existing micro fluorescent microscope generally adopts a light emitting diode as a light source, and needs to use a matched collimating lens and a matched narrow-band filter (such as Chinese patent application numbers 201510220640.8, 201680077967.7 and 201910621446.9), and the design has small volume and light weight, and can meet the basic requirement of in vivo imaging. But the observation range is small, the imaging resolution is low, and the focusing is very inconvenient. The most limited drawback is poor scalability, and it is difficult to change/increase the wavelength of light according to actual research needs. Particularly, if the light wavelength is increased to perform multicolor imaging or increase the optogenetic function, each time one light wavelength is increased, a set of light source consisting of a light emitting diode and a narrow-band filter needs to be added, and a dichroic mirror for guiding the light source needs to be additionally added, so that the size of the mirror body is increased, the weight is increased, the assembly is more complex, and the mirror body is not suitable for being worn on the head of an animal to continuously perform experiments. In view of the technical shortcomings, the research group has proposed a technical solution of simplifying the illumination light path and expanding the light emitting capability of the light source by externally arranging the light source of the imaging system through the multi-fiber coupled light beam arranged in a mixed manner (chinese patent application No. 201911299007.7). Because the technical scheme still adopts the wide-field fluorescence imaging technology, the technical scheme has the problem of low image contrast inherent in the wide-field fluorescence microscope. In order to solve this problem, other imaging methods with higher contrast ratio may be adopted, for example, chinese patent 201710183353.3 and chinese patent 201811494384.1 propose micro two-photon microscope solutions to improve the resolution and contrast ratio of imaging, however, this design must use a femtosecond pulse laser and a scanning control device, the hardware cost is very high, the structure is complex, and the imaging speed is slow due to the point scanning method.

The dynamic speckle illumination microscopic imaging is to use a random speckle pattern to illuminate a fluorescent object to acquire a series of images, and to obtain an optical tomography image by calculating the contrast of the images fluctuating along with time. Speckle is a granular light intensity distribution pattern and has the characteristic of high contrast; the fluorescence image obtained by illumination of the speckle pattern therefore also has a contrast, from which the information of the sample at the focal plane part can be obtained: high frequency information dominates at the focal plane while low frequencies dominate at off-focus positions. The observed speckle contrast corresponds to the ratio of focal plane to defocus information in the fluorescence image. The Mertz research group improves dynamic speckle illumination microscopic imaging, provides a method for mixing speckle and uniform illumination (called HiLo, Hi stands for high frequency, Lo stands for low frequency), only needs two times of imaging, and can obtain an optical tomography image through algorithm calculation: and calculating the local contrast of the speckle image and the uniform illumination image to obtain low-frequency focal plane information in the wide-field uniform illumination information, and combining the high-frequency information in uniform illumination to obtain the full-resolution optical tomography image through fusion.

The literature (optical fiber-based mixed illumination optical tomography fluorescence microscopy imaging research, DOI: 2.1016.779685) discloses a method for guiding light by utilizing multimode optical fibers and dissipating spots by utilizing vibration optical fibers, so that an optical fiber-based mixed illumination optical tomography fluorescence microscopy imaging system is realized, and the tomography capability of wide-field fluorescence microscopy imaging is improved.

It does not address the relationship between speckle illumination and the image, how the speckle particles affect the quality of the final image, and how the size of the speckles can be controlled to achieve the desired effect. There is no report about using the HiLo method to improve the image contrast in a head-mounted micro fluorescence microscope, and when the method is applied to a brain cell imaging device for a conscious and freely moving animal, because the imaging device has a smaller volume and a more complex illumination light path, the performance of speckle has a great influence on imaging: if the speckle particles are too large, the improvement of the contrast of the image processed by the HiLo algorithm is not obvious, and the aim of improvement cannot be achieved; if the speckle particles are too small, the processed image can produce artifacts, distorting the image.

Disclosure of Invention

Aiming at the defects or the improvement requirements of the prior art, the invention provides a fluorescent imaging illumination device, an imaging system and an imaging method based on the speckle principle, and aims to improve the contrast of tomography and avoid artifacts by controlling the quality and the quantity of speckle particles in a sampling window during speckle illumination, thereby obtaining a higher-quality tomography image and solving the technical problem of low image contrast in a miniature and wearable fluorescent microscopic imaging device in the prior art.

To achieve the above object, according to one aspect of the present invention, there is provided a fluorescence imaging illumination device based on the speckle principle, which includes a coupled fiber bundle, and one or more mixed excitation light generating units;

the coupled fiber bundle comprises a plurality of sub-bundles;

the mixed excitation light generating unit is used for generating coherent fluorescence excitation light and incoherent fluorescence excitation light with the same wavelength; the coherent fluorescence excitation light and the incoherent fluorescence excitation light are coupled to an illumination light path of the fluorescence imaging system through the same or different sub-beams of the coupling optical fiber bundle, the incoherent fluorescence excitation light forms shimming illumination, and the coherent fluorescence excitation light forms speckle illumination.

Preferably, the speckle particles of the speckle illumination satisfy the following relationship: the number of speckle particles in a single sampling window is 2-81, i.e.,

/Δs∈[1.4，9]

the sampling window is a minimum calculation area which is taken when the local contrast of the image is calculated by adopting a mixed speckle uniform illumination optical tomography algorithm, and is the size (side length) of the sampling window, and deltas is the size (diameter) of speckle particles.

Preferably, the numerical aperture of the optical fiber bundle is more than or equal to 0.5.

Preferably, the coupling fiber bundle of the fluorescence imaging illumination device is composed of a plurality of multimode fibers, preferably plastic fibers.

Preferably, the mixed excitation light generating unit further comprises a mode filter for filtering the mode of light transmitted in the optical fiber bundle, and the mode filter filters out the low-order mode and retains the high-order mode.

Preferably, the mixed excitation light generation unit of the fluorescence imaging illumination device includes a coherent fluorescence excitation light source and an incoherent fluorescence excitation light source, the coherent fluorescence excitation light source is preferably a laser, the incoherent fluorescence excitation light source is preferably a high-power LED-narrowband filter combination, and the coherent fluorescence excitation light source and the incoherent fluorescence excitation light source are coupled to the illumination light path through different sub-beams of a coupling optical fiber bundle or coupled to the illumination light path through the same sub-beam after being combined by the beam combining device; the beam combining device is preferably an optical fiber beam combiner or a space beam combining device.

Preferably, the fluorescence imaging illumination device, the mixed excitation light generation unit thereof, includes a coherent fluorescence excitation light source and a speckle attenuation device, the coherent fluorescence excitation light source and the speckle attenuation device are connected in series, and are coupled to the illumination light path through a sub-beam, that is, the speckle attenuation device is arranged at the light exit, the conduction optical fiber, and behind the optical fiber exit end; when the speckle attenuation device is started, the speckle effect is inhibited, and uniform light illumination spots are formed; when the speckle reduction device is turned off, speckle illumination is created.

According to another aspect of the present invention, there is provided an optical tomography image acquisition system based on the speckle principle, which includes the fluorescent imaging illumination device based on the speckle principle, a mirror body, an imaging device, and an acquisition control device provided by the present invention;

the fluorescence imaging illumination device generates illumination light to be projected to a target area through the mirror body, and the excited fluorescence is collected and imaged by the imaging device through the mirror body;

the acquisition control device is used for controlling the fluorescence imaging illumination device to alternately generate speckle illumination and uniform light illumination, and controlling the imaging device to perform speckle illumination imaging and uniform light illumination imaging.

Preferably, the mirror body of the optical tomography image acquisition system based on the speckle principle comprises a dichroic mirror, an objective lens, a fluorescent filter and an imaging lens; a dichroic mirror and an objective lens are sequentially arranged on the illumination light path; the imaging light path is sequentially provided with an objective lens, a dichroic mirror, a fluorescent filter and an imaging lens; the microscope body is a micro fluorescent microscope, is worn on the body of an experimental animal during work and is fixed at a part needing imaging;

the illumination light generated by the illumination device is transmitted to a dichroic mirror in the mirror body through a coupling fiber bundle, and is projected to a target area through an objective lens after being reflected; the fluorescence emitted by the target area is collected by the same objective lens and transmitted through the dichroic mirror, and is imaged to the image acquisition device by the imaging lens to form fluorescence image information.

Preferably, the optical path from the light outlet end of the optical fiber to the rear end face of the objective lens is 2 times of the focal length of the objective lens.

According to another aspect of the present invention, there is provided an imaging method using the optical tomography image acquisition system based on the speckle principle, which comprises the following steps:

(1) the fluorescence imaging lighting device of the system is controlled to alternately generate fluorescence excitation light for forming speckle illumination and fluorescence excitation light for forming uniform illumination, and the imaging device is controlled to perform speckle illumination imaging and uniform illuminationBright imaging, obtaining a sequence L of speckle-illuminated images of the same size_sAnd uniform light illumination imaging L_uThe image sequence of (1);

(2) illuminating each i of the image sequence imaged by the speckle illumination with the same size obtained in the step (1) according to the closest principle that the imaging phase difference time does not exceed the preset threshold value_sAnd each i of the image sequence imaged by the uniform light illumination_uPerforming correlation to obtain a speckle illumination imaging and uniform light illumination imaging correlation image pair sequence;

(3) correlating speckle illumination imaging and uniform illumination imaging obtained in step (2) with each image sequence pair (i)_s，i_u) And obtaining an optical chromatogram by adopting a HiLo optical chromatogram algorithm.

Preferably, the imaging method, step (1) thereof is a method of repeating the following steps a1, a 2; or repeating the following steps B1 and B2;

A1. the acquisition control device sends a trigger signal to a coherent light source in the mixed excitation light generation unit to generate coherent fluorescence excitation light, and simultaneously or after waiting for delay, sends the trigger signal to an image acquisition device in the endoscope body, the image acquisition device starts to acquire signals, and the delay length is less than the difference between the duration time of the coherent fluorescence excitation light and the acquisition time of the image acquisition device; the coherent fluorescence excitation light is transmitted to the lens body through the coupling optical fiber bundle, speckle illumination is formed at the focal plane of the objective lens, and at the moment, the image acquisition device acquires an image i of the speckle illumination_s(ii) a After the acquisition is finished, the acquisition control device sends a trigger signal to close the coherent light source;

A2. the acquisition control device sends a trigger signal to an incoherent light source in the mixed excitation light generation unit to generate an incoherent fluorescence excitation light source, and simultaneously or after waiting for delay, sends a trigger signal to the image acquisition device, the image acquisition device starts to acquire signals, and the delay length is less than the difference between the duration time of the incoherent fluorescence excitation light and the acquisition time of the image acquisition device; the incoherent fluorescence excitation light is transmitted to the lens body through the coupling optical fiber bundle, and uniform light illumination is formed at the focal plane of the lens body, and the image acquisition device acquires a uniform light illuminationImage i_u(ii) a After the acquisition is finished, the acquisition control device sends a trigger signal to close the incoherent light source;

B1. the acquisition control device sends a trigger signal to a coherent light source in the mixed excitation light generation unit to generate a coherent fluorescence excitation light source, and simultaneously or after waiting for delay, sends a trigger signal to an image acquisition device in the endoscope body, the image acquisition device starts to acquire signals, and the delay length is less than the difference between the duration time of the coherent fluorescence excitation light and the acquisition time of the image acquisition device; the light emitted by the light source is transmitted to the lens body through the coupling optical fiber bundle, the speckle illumination is formed at the focal plane of the objective lens, and at the moment, the image acquisition device acquires an image i of the speckle illumination_s；

B2. The acquisition control device sends a trigger signal to the speckle attenuating device to enable the speckle attenuating device to start to operate, and simultaneously or after waiting for delay, sends the trigger signal to the image acquisition device, the image acquisition device starts to acquire signals, and the delay length is less than the difference between the operating time of the speckle attenuating device and the acquisition time of the image acquisition device; at the moment, the light is transmitted to the lens body through the coupling optical fiber bundle and is homogenized by the speckle attenuation device, uniform light illumination is formed at the focal plane of the objective lens, and at the moment, the image acquisition device acquires an image i illuminated by the uniform light_u。

In general, compared with the prior art, the above technical solutions of the present invention can achieve the following advantages due to the provision of the fluorescent imaging illumination device, the imaging system and the imaging method based on the speckle principle.

(1) The invention selects the wearable fluorescent microscope system with an expandable light source, matches with various types of light sources such as a mixed coherent light source and an incoherent light source, obtains the optical tomography image of the target sample through algorithm processing, can greatly improve the contrast and resolution of imaging, and expands the application range of the wide-field fluorescent microscope system.

(2) The preferred embodiment of the invention takes full advantage of the inherent properties of optical fibers: the coherent light generates speckle effect after passing through the multimode fiber, and controls the diameter and the number of speckles, thereby realizing the purpose of improving the image contrast, having simple structure and low modification cost, and not increasing extra speckle control devices.

(3) The wearable microscope is durable and does not influence the wearing of small animals by improving the optical fiber bundle without changing any structure and size of the lens body part, and is very convenient and easy to realize.

Drawings

FIG. 1 is a schematic diagram of a hybrid illumination micro fluorescence imaging system according to the present invention;

FIG. 2 is a schematic cross-sectional view of the mirror body 3;

FIG. 3 is a schematic diagram of the structural connection of the hybrid light generating unit 1 and the coupling optical fiber bundle 2 in embodiment 1;

FIG. 4 is a schematic diagram of the structural connection of the hybrid light generating unit 1 and the coupling optical fiber bundle 2 in embodiment 2;

fig. 5 is a schematic diagram of a timing signal sent by the acquisition control unit 4 in embodiment 3;

FIG. 6 is a schematic diagram of the structural connection of the hybrid light generating unit 1 and the coupling optical fiber bundle 2 in embodiment 4;

fig. 7 is a schematic diagram of a timing signal sent by the acquisition control unit 4 according to embodiment 4.

Fig. 8 is a schematic structural connection diagram of the hybrid light generating unit 1 and the coupling optical fiber bundle 2 in embodiment 5.

FIG. 9 is a schematic view of the speckle reduction apparatus and the structural connection of the coupling fiber bundle 2 in embodiment 6;

fig. 10 is a schematic diagram of a timing signal sent by the acquisition control unit 4 in embodiment 6;

FIG. 11 is a schematic view showing the connection structure of the speckle reduction apparatus and the coupling fiber bundle 2 according to embodiment 7;

fig. 12 is a schematic structural connection diagram of the speckle reduction apparatus and the coupling fiber bundle 2 in embodiment 8.

The same reference numbers will be used throughout the drawings to refer to the same or like elements or structures, wherein:

1 is a mixed excitation light generating unit, S1 is a light genetic stimulation laser generating unit, 2 is a coupled fiber bundle, 3 is a mirror body, 4 is an acquisition control unit, 5 is a dichroic mirror, 6 is an objective lens, 7 is a fluorescent filter, 8 is an imaging lens 8, 9 is an image acquisition device, 10 is a laser, 101 is a driving module of the laser 10,11 is a high-power LED-narrow-band filter combination, 111 is a driving module of 11, 12 is a light genetic stimulation laser and 121 is a driving module of 12, 13 is an optical fiber beam combiner, 14 is an FC connector, 18 is a rigid sleeve, 202,203 and 204 are sub-beams of the coupled fiber bundle 2, 201 is a light outlet end of the coupled fiber bundle 2,20 is a diaphragm, 01 is a first mixed excitation light generating unit, 02 is a second mixed excitation light generating unit, 50 is a 470nm laser, 501 is a driving module of 50, 51 is a 470nm high-power LED-narrow-band filter combination, 511 is a 51 drive module 511, 52 is a 560nm laser, 521 is a 52 drive module; 53 is 560nm high-power LED-narrow-band filter combination, and 531 is 53 driving module. 912 is the speckle attenuation device, 913 is the lens, 921 is the ground glass piece, 922 is the step motor, 923 is the piezoelectric transducer.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The invention provides a fluorescent imaging lighting device based on a speckle principle, which comprises a coupling optical fiber bundle and one or more mixed excitation light generating units, and preferably further comprises an optogenetic stimulation laser generating unit;

the coupled fiber bundle comprises a plurality of sub-bundles; the coupling fiber bundle is preferably composed of a plurality of plastic fibers, preferably PMMA plastic fibers. Because the process of generating uniform light illumination by the light source of the invention can be accompanied with vibration or winding, the common glass optical fiber can generate mechanical fatigue and is easy to break and damage; the plastic optical fiber is light and flexible, is more resistant to damage (vibration and bending), and has the characteristics of excellent tensile strength and durability, so that each optical fiber in the coupling optical fiber bundle adopts the plastic optical fiber made of PMMA to adapt to the special application of the invention.

When the coupling optical fiber bundle is composed of a plurality of multimode optical fibers, the size of speckles is related to parameters of the multimode optical fibers and a mode of light transmission in the multimode optical fibers, so the mixed excitation light generating unit further comprises a mode screening device for screening the mode of light transmission in the optical fiber bundle, the mode screening device filters a low-order mode and reserves a high-order mode, smaller speckle particles are formed, and the effect of speckle illumination is improved.

As a preferred solution, the speckle particles of the speckle illumination satisfy the following relationship: the number of speckle particles in a single sampling window is 2-81; and satisfies the following conditions:

/Δs∈[1.4，9]

the sampling window is a minimum calculation area which is taken when the local contrast of the image is calculated by adopting a mixed speckle uniform illumination optical tomography algorithm, the minimum calculation area is the side length of the sampling window, and Delta s is the diameter of speckle particles.

The numerical aperture of the optical fiber bundle is more than or equal to 0.5.

As a preferred scheme, the mixed excitation light generation unit includes a coherent fluorescence excitation light source and an incoherent fluorescence excitation light source, the coherent fluorescence excitation light source is preferably a laser, the incoherent fluorescence excitation light source is preferably a high-power LED-narrowband filter combination, and the coherent fluorescence excitation light source and the incoherent fluorescence excitation light source are coupled to an illumination light path through different sub-beams of a coupling optical fiber bundle or coupled to the illumination light path through the same sub-beam after being combined by a beam combining device; the beam combining device is preferably an optical fiber beam combiner or a space beam combining device.

As a preferred scheme, the mixed excitation light generating unit comprises a coherent fluorescence excitation light source and a speckle attenuating device, wherein the coherent fluorescence excitation light source and the speckle attenuating device are connected in series and coupled into an illumination light path through a sub-beam, namely the speckle attenuating device is arranged at the light emergent position, the conducting optical fiber position and the rear end of the optical fiber emergent end; when the speckle attenuation device is started, the speckle effect is inhibited, and uniform light illumination spots are formed; when the speckle reduction device is turned off, speckle illumination is created.

The optical genetic spine laser generating unit generates optical genetic spine laser which is coupled to the illumination light path through the sub-beam of the coupling optical fiber bundle.

In the HiLo algorithm, the local speckle contrast, which in turn is determined by/Δ s, represents the square root of the number of speckle grains in each sampling window, determines the characteristics of the final image. That is, the square root of the number of speckle grains in each sampling window determines the characteristics of the final image, such as contrast. Through a plurality of experiments, the final image has better image contrast and no obvious artifact when the image is between 1.4 and 9.

The invention provides an optical tomography image acquisition system based on a speckle principle, which comprises a fluorescence imaging illumination device based on the speckle principle, a mirror body, an imaging device and an acquisition control device, wherein the fluorescence imaging illumination device is provided with a plurality of lenses;

the lens body comprises a dichroic mirror, an objective lens, a fluorescent filter and an imaging lens; a dichroic mirror and an objective lens are sequentially arranged on the illumination light path; the imaging light path is sequentially provided with an objective lens, a dichroic mirror, a fluorescent filter and an imaging lens; the microscope body is a micro fluorescent microscope, is worn on the body of an experimental animal during work and is fixed at a part needing imaging.

The illumination light generated by the illumination device is transmitted to a dichroic mirror in the mirror body through a coupling fiber bundle, and is projected to a target area through an objective lens after being reflected; the fluorescence emitted by the target area is collected by the same objective lens and transmitted through the dichroic mirror, and is imaged to the image acquisition device by the imaging lens to form fluorescence image information. According to the optical fiber transmission and light interference principle, when coherent fluorescence excitation light is transmitted in a multimode optical fiber, a speckle effect is formed at the emergent end of the optical fiber due to interference among different modes, while incoherent fluorescence excitation light cannot interfere, and therefore after the coherent fluorescence excitation light is transmitted through the multimode optical fiber, uniform illumination light spots can be formed at the emergent end.

And the optical path from the light outlet end of the optical fiber to the rear end face of the objective lens is at the focal length 2 times of the objective lens. Considering smaller device volume, the distance from the light-emitting end of the coupling optical fiber bundle to the rear end face of the objective lens is within 2.5 times of the focal length; however, when the distance is greater than 2 times of focal length, the light spot emitted by the optical fiber is reduced at the sample, and when the distance exceeds 2.5 times of focal length, the illumination range is reduced, the requirement of the visual field cannot be met, and when the distance is less than 2 times of focal length, the light spot emitted by the optical fiber is enlarged at the sample, which causes the speckle particle to be enlarged at the sample, thereby affecting the effect of the HiLo algorithm.

The imaging method of the optical tomography image acquisition system provided by the invention comprises the following steps:

(1) controlling a fluorescence imaging lighting device of the system to alternately generate fluorescence excitation light for forming speckle illumination and fluorescence excitation light for forming uniform light illumination, controlling the imaging device to perform speckle illumination imaging and uniform light illumination imaging, and obtaining an image sequence L of the speckle illumination imaging with the same size_sAnd uniform light illumination imaging L_uThe image sequence of (1); preferably:

repeating the following steps A1, A2:

A1. the acquisition control device sends a trigger signal to a coherent light source in the mixed excitation light generation unit to generate coherent fluorescence excitation light, and simultaneously or after a delay, sends a trigger signal to an image acquisition device in the endoscope body, the image acquisition device starts to acquire signals, and the delay is used for delaying the generation of the coherent fluorescence excitation lightThe delay length is smaller than the difference between the duration of the coherent fluorescence excitation light and the acquisition time of the image acquisition device, for example, several milliseconds. The coherent fluorescence excitation light is transmitted to the lens body through the coupling optical fiber bundle, speckle illumination is formed at the focal plane of the objective lens, and at the moment, the image acquisition device acquires an image i of the speckle illumination_s(ii) a And after the acquisition is finished, the acquisition control device sends a trigger signal to close the coherent light source.

A2. The acquisition control device sends a trigger signal to an incoherent light source in the mixed excitation light generation unit to generate an incoherent fluorescence excitation light source, and simultaneously or after waiting for delay, sends a trigger signal to the image acquisition device, and the image acquisition device starts to acquire signals, wherein the delay length is less than the difference between the duration time of the coherent fluorescence excitation light and the acquisition time of the image acquisition device, such as several milliseconds. The incoherent fluorescence excitation light is transmitted to the lens body through the coupling optical fiber bundle, uniform light illumination is formed at the focal plane of the lens body, and at the moment, the image acquisition device acquires an image i illuminated by the uniform light_u(ii) a And after the acquisition is finished, the acquisition control device sends a trigger signal to close the incoherent light source.

Or repeating the following steps B1, B2:

B1. the acquisition control device sends a trigger signal to a coherent light source in the mixed excitation light generation unit to generate a coherent fluorescence excitation light source, and simultaneously or after waiting for delay, sends a trigger signal to an image acquisition device in the endoscope body, the image acquisition device starts to acquire signals, and the delay length is less than the difference between the duration time of the coherent fluorescence excitation light and the acquisition time of the image acquisition device, such as a plurality of milliseconds. The light emitted by the light source is transmitted to the lens body through the coupling optical fiber bundle, the speckle illumination is formed at the focal plane of the objective lens, and at the moment, the image acquisition device acquires an image i of the speckle illumination_s；

B2. The collecting control device sends a trigger signal to the speckle attenuating device to enable the speckle attenuating device to start to operate, and simultaneously or after waiting for delay, sends the trigger signal to the image collecting device to enable the image collecting device to start to collect signals, wherein the delay length is smaller than the difference between the operating time of the speckle attenuating device and the collecting time of the image collecting device. At this time, the light is transmitted to the mirror body through the coupling optical fiber bundle,and homogenized by the speckle attenuating device to form uniform light illumination on the focal plane of the objective lens, and the image acquisition device acquires an image i illuminated by the uniform light_u；

The illumination duration time of the coherent fluorescent agent light-emitting light source and the incoherent fluorescence excitation light source is generally far longer than several milliseconds, so that the delay is increased, the unstable illumination time period in the light generation stage is avoided, and a better image acquisition effect is achieved.

(2) According to the principle that the phase difference time does not exceed a preset threshold value, illuminating and imaging each i of the image sequence of the speckle illumination imaging with the same size obtained in the step (1)_sAnd each i of the image sequence imaged by the uniform light illumination_uPerforming correlation to obtain a speckle illumination imaging and uniform light illumination imaging correlation image pair sequence; the preset threshold value does not exceed one imaging period.

(3) Correlating speckle illumination imaging and uniform illumination imaging obtained in step (2) with each image sequence pair (i)_s，i_u) Obtaining an optical tomography image i by using an optical tomography algorithm (Hilo algorithm)_HiLo(ii) a Real-time display and/or storage can be selected, and due to the fact that the calculation speed is very high, the effect of almost real-time display can be achieved; alternatively, the images acquired only during a certain period of time of interest may be algorithmically processed and displayed in real time or/and saved. In particular optical tomographic image i_HiLo：

i_HiLo＝i_HP+ηi_LP

Wherein eta is obtained by calculating the weight by ensuring that the optical transfer function between high and low frequencies does not generate mutation, and is generally 1; i.e. i_HPInformation of the high-frequency part of the focal plane, i_LPInformation of a low-frequency part of a focal plane;

information i of the low frequency part of the focal plane_LPThe method comprises the following steps:

i_LP＝F^-1{I_su×LP}

wherein, I_suIs i_suFourier transform of (i)_su＝Ci_u，CIs partially emptyContrast between the two; LP is a Gaussian low-pass filter with a cut-off frequency of k_c，k_cIs taken as

Wherein the window length of the local space.

The local spatial contrast CThe calculation method of (2) is as follows:

wherein std (i)_{d_BP})And μ (i)_{d_BP})Respectively represent i_{d_BP}Standard deviation and mean, i, in a window of length_{d_BP}As a difference image i_dFiltering with Gaussian band-pass filter to obtain difference image i_d＝i_s-i_uThe Gaussian band-pass filter is

Information of high frequency part of the focal plane i_HPImage i imageable by uniform illumination_uObtaining, as follows:

i_HP＝F^-1{I_u×HP}

wherein, I_uImage i imaged for over-uniform light illumination_uThe fourier transform of (a), HP is a high-pass filter complementary to LP, HP + LP is 1, F^-1Is an inverse fourier transform.

The following are examples:

example 1 in situ optogenetic-monochromatic fluorescence imaging System

The following takes the in-situ optogenetic-monochromatic fluorescence imaging application as an example to describe in detail an improved micro fluorescence imaging system provided by the present invention, as shown in fig. 1 and fig. 2, comprising a fluorescence imaging illumination device, a mirror body 3 and an acquisition control unit 4

The lens body 3 is fixed on a part needing imaging on a target, such as a head, and the lens body 3 is a micro fluorescence microscope and comprises a dichroic mirror 5, an objective lens 6, a fluorescence filter 7, an imaging lens 8 and an image acquisition device 9.

The fluorescence imaging illumination device comprises: a mixed excitation light generation unit 1, a photoexcitation laser generation unit S1, and a coupling fiber bundle 2.

The detailed structure and connection method of the mixed excitation light generation unit 1 and the coupling fiber bundle 2 are shown in fig. 3. The mixed excitation light generation unit 1 includes: the laser 10 and the driving module 101 thereof, the laser 10 emits the fluorescence excitation light with 450nm, and the output power can reach 50 mw; the high-power LED-narrowband filter combination 11 and the driving module 111 thereof emit fluorescence excitation light with the wavelength of 450 nm;

the light emitted by the laser 10 and the LED-narrowband filter assembly 11 is combined into one path of light by the optical fiber combiner 13, and the other end of the combiner is connected to one sub-beam 202 of the coupling optical fiber bundle 2 by the FC connector 14. If the laser or the LED outputs spatial light, all the fiber coupling modes are replaced by corresponding spatial light coupling modes, which are common knowledge in the field and will not be described herein again. The mixed excitation light generating unit 1 further comprises a diaphragm 20 (not shown in the figure) installed at the light exit hole of the laser 10, the diaphragm 20 is a disc or annular blocking target with a diameter of 50-100um, the diaphragm 20 is used as a mode filter to filter out low-order modes and retain high-order modes in the optical fiber bundle, so that the particles are smaller during speckle illumination.

The optogenetic stimulation laser generating unit S1 includes: the laser 12 and the driving module 121 thereof, the laser 12 emits 633nm stimulating light, and the power can reach 100 mw. Both the laser and the LED have a standard FC interface for connection with a coupling fiber bundle 2.

The coupling optical fiber bundle 2 comprises 19 plastic optical fibers made of PMMA, each plastic optical fiber has an outer diameter of 125um, a numerical aperture of 0.5 and a length of 2 meters, and is closely arranged at a light-emitting end 201 and divided into two

sub-bundles

202 and 203 at a light-entering end; the sub-beam 202 is connected to one end of the optical combiner 13 via a standard FC connector 14, and the sub-beam 203 is connected to the laser 12 via a standard FC interface. Beamlet 202 comprises 10 fibers distributed as shown in cross-section B-B16, and beamlet 203 comprises 9 fibers distributed as shown in cross-section C-C17. The optical fibers of the sub-beams 202 and 203 are arranged in a mixed manner in the light exit end 201, as shown in cross section a-a15, to ensure that the exit spots of the light from different sub-beams at the light exit end 201 coincide. The distance from the light outlet end 201 of the coupling optical fiber bundle 2 to the rear end face of the objective lens 6 is within 2.5 times of the focal length, the diameter of the coupling optical fiber bundle 2 is 0.625 mm, and the densities of the optical fibers of the sub-bundles 202 and 203 are respectively 51 and 46 fibers/square mm. The light-emitting end 201 of the coupling optical fiber bundle 2 is wrapped by a section of rigid sleeve 18 and fixed at the near-dichroic mirror 5 through AB glue and is positioned at the 2-time focal length of the rear end face of the objective lens 6.

The lens body 3 is filled with a polyformaldehyde resin material and is used for setting a light path, and the lens body 3 is sequentially provided with an objective lens 6, a dichroic mirror 5, a fluorescent light filter 7, an imaging lens 8 and an image acquisition device 9 along a fluorescent imaging light path. The diameter of the objective lens 6 is 1.8mm, and the focal length is 1.71 mm; the size of the dichromatic mirror 5 is 5 × 4 × 1mm, the dichromatic mirror transmits light at 500-; the fluorescence filter 7 is a 500-550nm transmission band-pass fluorescence filter; the diameter of the imaging lens 8 is 5mm, the focal length is 10mm, and the optical magnification of the system is 5.85 times; and the optical path from the light outlet end of the optical fiber to the rear end face of the objective lens is at the focal length 2 times of the objective lens.

The driving modules are all connected with the acquisition control unit 4, and the acquisition control unit 4 sends out TTL signals to control the light emission of the TTL signals 10,11 and 12 according to experimental requirements.

The image acquisition device 9 comprises an array image detector and a peripheral circuit thereof, wherein the array image detector is a CMOS and is connected with the acquisition control unit 4 through a high-speed interface, the acquisition rate can reach more than 30 frames/second, and the acquisition starting time of each frame of image is output to the acquisition control unit 4 in a pulse form for acquisition and recording; the acquisition control unit 4 can communicate with an upper computer (computer), and the TTL pulse with fixed frequency and duty ratio is edited by software and is output to the mixed excitation light generation unit 1 by the acquisition control unit 4, so that modulation of the laser 12 of the stimulus laser source and time sequence control of the laser 10 and the LED-narrowband filter combination 11 are realized.

The imaging process can be described as: at a certain moment, the 450nm fluorescence excitation light emitted by the laser 10 or the high-power LED-narrowband filter combination 11 and the 633nm optical excitation laser emitted by the laser 12 in the mixed excitation light generating unit 1 are respectively coupled to the

corresponding sub-beams

202 and 203, and are simultaneously transmitted to the dichroic mirror 5 in the mirror body 3 by the coupling optical fiber bundle 2, and are projected to a target area through the objective lens 6 after being reflected; the neuron activity of the target area is accompanied by the emission of fluorescence, and the fluorescence is collected by the same objective lens 6, transmitted through the dichroic mirror 5 and imaged by the imaging lens 8 to the image acquisition device 9 to form fluorescence image information.

Example 2 in situ optogenetic-Monochromatic fluorescence imaging System

The following is another implementation scheme of in-situ optogenetic-monochromatic fluorescence imaging application, so as to illustrate an improved micro fluorescence imaging system provided by the present invention, most of the structure of which is the same as that of the system in example 1, and the description of the same places is omitted, and only the places different from example 1 are described here.

The system is shown in fig. 1 and fig. 2, and all the parts are the same as the embodiment 1, except that:

the detailed structure and connection of the mixed excitation light generating unit 1 and the coupling fiber bundle 2 of the fluorescence imaging illumination device are shown in fig. 4.

The mixed excitation light generation unit 1 includes: the laser 10 and the driving module 101 thereof, the laser 10 emits the fluorescence excitation light with 450nm, and the output power can reach 50 mw; the high-power LED-narrowband filter combination 11 and the driving module 111 thereof emit fluorescence excitation light with the wavelength of 450 nm;

both the laser and the LED have a standard FC interface for connection with a coupling fiber bundle 2. The driving modules are all connected with the acquisition control unit 4, and the acquisition control unit 4 sends out TTL signals to control the light emission of the TTL signals 10,11 and 12 according to experimental requirements. The mixed excitation light generating unit 1 further comprises a diaphragm 20 (not shown in the figure) installed at the light exit hole of the laser 10, the diaphragm 20 is a disc or annular blocking target with a diameter of 50-100um, the diaphragm 20 is used as a mode filter to filter out low-order modes and retain high-order modes in the optical fiber bundle, so that the particles are smaller during speckle illumination.

The optogenetic stimulation laser generating unit S1 includes: the laser 12 and the driving module 121 thereof, the laser 12 emits 633nm stimulating light, and the power can reach 100 mw.

The coupling optical fiber bundle 2 comprises 19 plastic optical fibers made of PMMA, each plastic optical fiber is 125 microns in outer diameter, 0.5 in numerical aperture and 2 meters in length, the plastic optical fibers are closely arranged at a light outlet end 201 and are divided into three sub-bundles 202,203 and 204 at a light inlet end; the sub-beam 202 is connected to the laser 10 via a standard FC interface, the sub-beam 203 is connected to the high power LED-narrowband filter assembly 11 via a standard FC interface, and the sub-beam 204 is connected to the laser 12 via a standard FC interface. Beamlet 202 comprises 1 fiber, whose distribution is shown in cross-section B-B16, beamlet 203 comprises 12 fibers, whose distribution is shown in cross-section C-C17, and beamlet 204 comprises 6 fibers, whose distribution is shown in cross-section D-D19. The optical fibers of the sub-beams 202,203 and 204 are arranged in a hybrid manner in the light exit end 201, as shown in cross-section a-a15, to ensure that the exit spots of the light from the different sub-beams at the light exit end 201 coincide. The distance from the light outlet end 201 of the coupling optical fiber bundle 2 to the rear end face of the objective lens 6 is within 2.5 times of the focal length, the diameter of the coupling optical fiber bundle 2 is 0.625 mm, and the densities of the optical fibers of the sub-bundles 202,203 and 204 are 20, 46 and 31 pieces/square mm respectively. The light-emitting end 201 of the coupling optical fiber bundle 2 is wrapped by a section of rigid sleeve 18 and fixed at the near-dichroic mirror 5 through AB glue and is positioned at the 2-time focal length of the rear end face of the objective lens 6.

The rest of the structure is the same as the system in embodiment 1, and is not described again.

Example 3 in situ optogenetic-Monochromatic fluorescence imaging method

The following describes an improved micro fluorescence imaging method provided by the present invention in detail with reference to the systems of examples 1 and 2.

The acquisition control unit 4 should be able to communicate with an upper computer (computer), the acquisition control unit 4 outputs TTL pulses with fixed frequency and duty ratio edited by software, and a timing signal sent by the acquisition control unit 4 is shown in fig. 5. The image capturing means 9 is set to start capturing an image at the rising edge by setting the laser 12, the laser 10 and the LED11 to be on at the rising edge and to remain on at a high level, to be off at the falling edge and to remain off at a low level. For simplicity of description, it is assumed that 633nm stimulation light is always kept in an on state during experiments, and during actual use, TTL pulses are edited to control the on-off time and intensity of the stimulation light according to optogenetic control requirements.

1. In the experimentThe collection control unit 4 sends TTL signals to the mixed excitation light generation unit 1 and the image collection device 9 at t₁At the moment, the

lasers

10 and 12 are turned on to emit fluorescence excitation light of 450nm and excitation light of 633nm respectively, the LED11 is kept turned off, and simultaneously or after a delay of several milliseconds, the image acquisition device 9 starts to acquire signals. At the moment, 450nm fluorescence excitation light emitted by the laser 10 forms speckle illumination in an imaging target area, and the image acquisition device 9 acquires a speckle illumination image I₁。

2. At t₂At that moment, the laser 10 is turned off when encountering a falling edge, and the LED11 is turned on when encountering a rising edge, and is kept for a while, or after waiting for a delay of several milliseconds, the image acquisition device 9 starts acquiring signals. At the moment, the 450nm fluorescence excitation light emitted by the LED11 forms uniform light illumination in the imaging target area, and the image acquisition device 9 acquires an image I illuminated by the uniform light illumination₂。

3. Step 1-2 is a period, step 1-2 is executed circularly through TTL signal control, and the image acquisition device 9 can obtain a series of images I with alternate speckle illumination and uniform light illumination₁，I₂，……I_nCalculating a chromatographic pattern P by using a mixed speckle uniform light illumination optical chromatographic algorithm for every two adjacent patterns₁，P₂，……P_n-1Real-time display and/or storage can be selected, and due to the fact that the calculation speed is very high, the effect of almost real-time display can be achieved; alternatively, the images acquired only during a certain period of time of interest may be algorithmically processed and displayed in real time or/and saved.

For image pair (I)₁，I₂)、(I₃，I₂)、(I₃，I_k)、(I₅，I_k)、……(I_n-1，I_n): obtaining a chromatographic chart P by adopting a mixed speckle uniform light illumination optical tomography algorithm₁，P₂，……P_n-1The method comprises the following steps:

each image sequence pair (i)_s，i_u) And obtaining an optical tomography image by adopting an optical tomography algorithm (Hilo algorithm): firstly, a speckle illumination image i is obtained_sAnd is uniformIlluminated image i_uTo find a difference image i_d＝i_s-i_uUsing a Gaussian band-pass filter

To i_dFiltering to obtain i_{d_BP}Definition of i_{d_BP}Local spatial contrast of (2):

wherein std (i)_{d_BP})And μ (i)_{d_BP})Respectively represent i_{d_BP}Standard deviation and mean in a window of length. Using contrast as weight value and uniform illumination image i_uMultiplication i_su＝Ci_uThen, the information of the low frequency part of the focal plane can be extracted:

i_LP＝F^-1{I_su×LP}

wherein, I_suIs i_suLP is a Gaussian low-pass filter with a cut-off frequency k_c，k_cIs taken as

HP is a high-pass filter complementary to LP, LP + HP ═ 1, F^-1Is an inverse fourier transform.

While the high-frequency part information of the focal plane can pass through i_uDirectly extracting:

i_HP＝F^-1{I_u×HP}

I_uis i_uThe fourier transform of (d).

And finally, fusing the high-frequency part information and the low-frequency part information to obtain the optical tomography image of the focal plane.

i_HiLo＝i_HP+ηi_LP

Wherein the eta value is obtained by calculation by ensuring that the optical transfer function between high and low frequencies does not generate mutation, and the value is 1.

Example 4 two-color fluorescence imaging System and method

An improved micro fluorescence imaging system is used for two-color fluorescence imaging, most of the structure of the improved micro fluorescence imaging system is the same as that of the system in the embodiment 1, the description of the same parts is omitted, and only the parts different from the embodiment 1 are described herein.

The system is shown in fig. 1 and fig. 2, and all the parts are the same as the embodiment 1, except that: the fluorescence imaging illumination device is shown in fig. 6 and comprises:

first mixed excitation light generation unit 01, including: 470nm laser 50 and its driving module 501; 470nm high-power LED-narrowband filter combination 51 and driving module 511 thereof; 560nm laser 52 and its driving module 521;

second mixed excitation light generation unit 02 includes: 560nm high power LED-narrowband filter combination 53 and its driving module 531.

The

driving module

501 and 531 are configured to modulate the light sources according to the external TTL signals, and light the two groups of light sources in a time-sharing manner in cooperation with the imaging frame frequency of the image capturing device 9.

470nm light emitted by the laser 50 and the LED-narrowband filter combination 51 is combined into one path of light through the first optical fiber beam combiner 13, and the other end of the beam combiner is connected with one sub-beam 202 of the coupling optical fiber beam 2 through the first FC connector 14; the 560nm light from the laser 52 and the LED-narrowband filter combination 53 is combined into one light beam by the second fiber combiner 13, and the other end of the combiner is connected to one sub-beam 203 of the coupling fiber bundle 2 by the second FC connector 14. If the laser or the LED outputs spatial light, all the fiber coupling modes are replaced by corresponding spatial light coupling modes, which are common knowledge in the field and will not be described herein again. The mixed excitation light generating unit 1 further includes a diaphragm (not shown) installed at the light exit holes of the

lasers

50 and 52, and the diaphragm is a disc or ring-shaped blocking target with a diameter of 50-100 um.

The coupling fiber bundle 2 comprises 37 plastic fibers made of PMMA, the numerical aperture is 0.5, the length is 2 meters, the diameter of each fiber is 0.125 mm, the fibers are closely arranged at the light-emitting end 201, and the fibers are divided into two

sub-bundles

202 and 203 at the light-entering end. Beamlet 202 comprises 19 optical fibers, distributed as shown in cross-section B-B16, and beamlet 203 comprises 18 optical fibers, distributed as shown in cross-section C-C17. The optical fibers of the sub-beams 202 and 203 are arranged in a mixed manner in the light exit end 201, as shown in cross section a-a15, to ensure that the exit spots of the light from different sub-beams at the light exit end 201 coincide. The light outlet end 201 of the coupling optical fiber bundle 2 is located at 1.8 times focal length of the rear end of the objective lens 6, the diameter of the coupled optical fiber bundle 2 is 0.875 mm, and the optical fiber densities of the sub-bundles 202 and 203 are 43 and 41 fibers/square mm respectively. The light-emitting end 201 of the coupling optical fiber bundle is wrapped by a section of rigid sleeve 18 and fixed with the fluorescence imaging optical path through AB glue.

Because the dual-wavelength excitation fluorescence is adopted, the fluorescence filter 7 in the mirror body 3 is a 500-550nm and 580-620nm dual-band-pass fluorescence filter, the dichroscope 5 transmits at 500-550nm and 580-620nm, and the rest visible light wave band is reflected at 45 degrees; the image acquisition device 9 comprises an array image detector and a peripheral circuit thereof, wherein the array image detector is a 480 × 752 gray scale array image detector.

The imaging process can be described as: the laser 50, the high-power LED-narrowband filter combination 51, the laser 52 and the high-power LED-narrowband filter combination 53 are sequentially lightened according to time sequence control, fluorescence excitation light is transmitted to the dichroic mirror 5 in the mirror body 3 through the coupling optical fiber bundle 2, and is projected to a target area through the objective lens 6 after being reflected; the neuron activity of the target area is accompanied by the emission of fluorescence, and the fluorescence is collected by the same objective lens 6, transmitted through the dichroic mirror 5 and imaged by the imaging lens 8 to the image acquisition device 9 to form fluorescence image information.

The following describes the method of two-color fluorescence imaging in detail with reference to the above system.

The acquisition control unit 4 should be able to communicate with an upper computer (computer), the acquisition control unit 4 outputs TTL pulses with fixed frequency and duty ratio edited by software, and a timing signal sent by the acquisition control unit 4 is shown in fig. 7. Setting the laser 50, the LED51, the laser 52, and the LED53 to be turned on at the rising edge and to remain turned on at a high level, and to be turned off at the falling edge and to remain turned off at a low level, the image capturing device 9 starts capturing an image at the rising edge.

1. During the experiment, the acquisition control unit 4 sends TTL signals to the mixed excitation light generating unit 1 and the image acquisition device 9, and at t₁At that moment, the laser 50 is turned on to emit 470nm fluorescence excitation light, the remaining light sources are kept off, and simultaneously or after a delay of several milliseconds, the image acquisition device 9 starts to acquire signals. At this time, 470nm fluorescence excitation light emitted by the laser 50 forms speckle illumination in the imaging target area, and the image acquisition device 9 acquires an image I of the 470nm speckle illumination₁。

2. At t₂At that moment, the laser 50 is turned off when encountering a falling edge, and the LED51 is turned on when encountering a rising edge, and is kept for a while, or after waiting for a delay of several milliseconds, the image acquisition device 9 starts acquiring signals. At the moment, 470nm fluorescence excitation light emitted by the LED51 forms uniform light illumination in an imaging target area, and the image acquisition device 9 acquires an image I illuminated by the 470nm uniform light illumination₂。

3. At t₃At that moment, the LED51 turns off when encountering a falling edge and the laser 52 turns on when encountering a rising edge, and remains on for a while, or waits for a delay of several milliseconds before the image capture device 9 begins capturing a signal. At the moment, 560nm fluorescence excitation light emitted by the laser 52 forms speckle illumination in an imaging target area, and the image acquisition device 9 acquires an image J of 560nm speckle illumination₁。

4. At t₄At that moment, the laser 52 is turned off when encountering a falling edge and the LED53 is turned on when encountering a rising edge, and is kept for a while, or after waiting for a delay of several milliseconds, the image acquisition device 9 starts acquiring signals. At the moment, 560nm fluorescence excitation light emitted by the LED53 forms uniform light illumination in an imaging target area, and the image acquisition device 9 acquires an image J illuminated by the 470nm uniform light illumination₂。

5. The steps 1-4 are a period, n periods are controlled and circularly executed through TTL signals, and the image acquisition device 9 can obtain a series of images I with alternative 470nm speckle illumination and uniform light illumination₁，I₂，……I_2nAnd a series of 560nm speckle illuminations and symmetriesImage J with alternate uniform illumination₁，J₂，……J_2n. For every two pictures I_mAnd I_m+1(m-1, 2,3, …, 2n-1) calculating 470nm optical chromatogram P by using mixed speckle uniform light illumination optical chromatogram algorithm₁，P₂，……P_n(ii) a For every two pictures J_mAnd J_m+1(M is 1,2,3, …, 2n-1) calculating 560nm optical chromatogram M by using mixed speckle homogeneous light illumination optical chromatogram algorithm₁，M₂，……M_n. Real-time display and/or storage can be selected, and due to the fact that the calculation speed is very high, the effect of almost real-time display can be achieved; alternatively, the images acquired only during a certain period of time of interest may be algorithmically processed and displayed in real time or/and saved.

Example 5 in situ optogenetic-two color fluorescence imaging system and method.

An improved micro fluorescence imaging system is used for in-situ optogenetic-bichromatic fluorescence imaging, most of the structure of the improved micro fluorescence imaging system is the same as that of the system in the embodiment 4, the description of the same parts is omitted, and only the parts different from the embodiment 4 are described herein.

The system is shown in fig. 1 and fig. 2, and all the parts are the same as the embodiment 4, and the differences are that: as shown in fig. 8, the fluorescence imaging illumination apparatus further includes a light genetic stimulation laser generation unit in addition to embodiment 4: a 633nm laser 12 and its driving module 121 for optogenetic stimulation.

Therefore, the coupling fiber bundle 2 needs to be divided into three sub-bundles 202,203 and 204 at the light-incoming end, the coupling fiber bundle 2 includes 37 plastic fibers, and the diameter of each fiber is 0.125 mm. Beamlet 202 comprises 13 fibers, the distribution of which is shown in cross-section B-B16, beamlet 203 comprises 12 fibers, the distribution of which is shown in cross-section C-C17, and beamlet 204 comprises 12 fibers, the distribution of which is shown in cross-section D-D19. The optical fibers of the three sub-beams are arranged in a mixed manner in the light exit end 201, as shown in the cross section a-a15, to ensure that the emergent spots of the light from different sub-beams at the light exit end 201 coincide. The light outlet end 201 of the coupled fiber bundle 2 is located at 2 times focal length at the rear end of the objective lens 6, the diameter of the coupled fiber bundle 2 is 0.875 mm, and the fiber densities of the sub-bundles 202,203 and 2/4 are 46/42 and 42 fibers/mm. The light-emitting end 201 of the coupling optical fiber bundle is wrapped by a section of rigid sleeve 18 and fixed with the fluorescence imaging optical path through quick-drying glue.

The imaging process is the same as that of embodiment 4, except that a 633nm laser 12 is added to the embodiment 4 as a light genetic stimulation light source, and is modulated by a fixed frequency and duty ratio TTL signal, and the TTL pulse signal comes from the acquisition control unit 4.

The in-situ optogenetic-bicolor fluorescence imaging method is completely the same as that in embodiment 4, except that the TTL signal of the laser 12 is added on the basis of embodiment 4, and in an actual experiment, the frequency and duty ratio of TTL pulses can be edited according to optogenetic control requirements, which is not described herein again.

Example 6 laser speckle attenuator, in-situ optogenetic-monochromatic fluorescence imaging System and method

In the following, a laser speckle attenuator (LSR) is taken as a speckle attenuator to implement in-situ optogenetic-monochromatic fluorescence imaging, and a detailed description is given to a hybrid illumination micro fluorescence imaging system provided by the present invention, most of the structure of which is the same as that in embodiment 1, and the description of the same parts is omitted, and only the parts different from embodiment 1 are described herein.

The detailed structure and connection of the speckle reduction device and the coupling fiber bundle 2 are shown in fig. 9. Its fluorescence imaging lighting device includes:

the mixed excitation light generation unit 1 includes: the laser 10 and the driving module 101 thereof, the laser 10 emits the fluorescence excitation light with 450nm, and the output power can reach 50 mw; the speckle attenuation device is an LSR912, light emitted by the laser 10 is converged by a first lens 913 and then incident on the LSR912, the light is modulated by the LSR912 and then collimated by a second lens 913 and then emitted, a third lens 913 is used for coupling a light beam into a coupled optical fiber beam, and 14 is an FC connector or an optical fiber coupler.

The optogenetic stimulation laser generating unit S1 includes: the laser 12 and the driving module 121 thereof, the laser 12 emits 633nm stimulating light, and the power can reach 100 mw. The lasers all have a standard FC interface for connection with a coupling fiber bundle 2.

The driving module and the LSR are both controlled by the acquisition control unit 4, and the acquisition control unit 4 sends out TTL signals to control the operation or the closing of the TTL signals 10,12 and 912 according to the experimental requirements.

The method of imaging is described in detail below in conjunction with the above system.

The acquisition control unit 4 should be able to communicate with an upper computer (computer), the acquisition control unit 4 outputs TTL pulses with fixed frequency and duty ratio edited by software, and a timing signal sent by the acquisition control unit 4 is shown in fig. 10. The laser 12, the laser 10 and the LSR912 are set to be turned on at a rising edge and to be kept in an on state at a high level, and to be turned off at a falling edge and to be kept in an off state at a low level, and the image capturing device 9 starts capturing an image at the rising edge. For simplicity of description, it is assumed that 633nm stimulation light is always kept in an on state during experiments, and during actual use, TTL pulses are edited to control the on-off time and intensity of the stimulation light according to optogenetic control requirements.

1. During the experiment, the acquisition control unit 4 sends TTL signals to the light-emitting unit 1 and the image acquisition device 9, and at t₁At the moment, the

lasers

10 and 12 are turned on to respectively emit fluorescence excitation light of 450nm and stimulation light of 633nm, the LSR912 is kept turned off, and simultaneously or after a delay of several milliseconds, the image acquisition device 9 starts to acquire signals. At this time, because the LSR912 is kept closed, the 450nm fluorescence excitation light emitted by the laser 10 directly passes through the LSR912 without being modulated, so that the laser forms speckle illumination in the imaging target area after passing through the coupling optical fiber bundle 2, and the image acquisition device 9 acquires an image I of the speckle illumination₁。

2. At t₂At the moment, the LSR912 opens when encountering the rising edge and keeps for a while, and at the same time or waits for a delay of several milliseconds before the image capturing device 9 starts capturing signals. At the moment, because the LSR912 is in a working state, the emitted fluorescence excitation light with 450nm is modulated by the LSR912 and then enters the coupling optical fiber bundle 2, the laser speckle effect is weakened, the laser forms uniform light illumination in an imaging target area, and the image acquisition device 9 acquires an image I illuminated by the uniform light illumination₂。

3. Step 1-2 is a period, and step 1-2 is executed circularly through TTL signal controlThe image acquisition device 9 can obtain a series of images I with alternate speckle illumination and uniform light illumination₁，I₂，……I_nCalculating a chromatographic pattern P by using a mixed speckle uniform light illumination optical chromatographic algorithm for every two adjacent patterns₁，P₂，……P_n-1Real-time display and/or storage can be selected, and due to the fact that the calculation speed is very high, the effect of almost real-time display can be achieved; alternatively, the images acquired only during a certain period of time of interest may be algorithmically processed and displayed in real time or/and saved.

Example 7 ground glass sheet

The vibrating or rotating ground glass sheet (diffuser sheet) may also be used as a speckle reduction device, and the following takes the rotating ground glass sheet as the speckle reduction device to implement in-situ optogenetic-monochromatic fluorescence imaging, which details an improved micro fluorescence imaging system provided by the present invention, and most of the structures of the improved micro fluorescence imaging system are the same as those in embodiment 6, and the same parts are not repeated herein, and only the parts different from embodiment 6 are described herein.

As shown in fig. 11, the speckle reduction device includes a ground glass sheet 921, a stepping motor 922, a power supply and a drive (not shown), wherein the ground glass sheet 921 is made of BK7 and has a granularity of 1500 meshes. The ground glass piece 921 is fixed on the stepping motor 922, and the stepping motor 922 can drive the ground glass piece 921 to rotate. The driving of the stepping motor 922 receives an instruction sent by the acquisition control unit 4, and controls the stepping motor 922 to rotate or stop rotating as required. In this embodiment, the maximum rotation speed is 468 rpm.

When the acquisition control unit 4 controls the stepping motor 922 to drive the ground glass piece 921 to rotate, laser irradiates on the rotating ground glass piece 921 to generate partial coherent light, so that the speckle effect can be inhibited, and at the moment, uniform light illumination is generated in a target area; when the acquisition control unit 4 controls the stepping motor 922 to stop rotating, speckle illumination is generated in the target area, so that two illumination modes are generated, and the specific imaging method is the same as that in embodiment 6, and is not described again.

EXAMPLE 8 piezoelectric transducer

The optical fiber is fixed on a vibrating motor or a piezoelectric transducer (PZT) and can also be used as a speckle reduction device, and the optical fiber is wound on the PZT to be used as the speckle reduction device to realize in-situ optogenetic-monochromatic fluorescence imaging, so as to describe in detail an improved micro fluorescence imaging system provided by the present invention, most of the structure of which is the same as that in embodiment 6, and the description of the same places is omitted, and only the places different from embodiment 6 are described herein.

As shown in fig. 12, the speckle reduction apparatus includes a piezoelectric transducer (PZT)923, a power supply and a driver (not shown), wherein the driving frequency of the PZT923 is 0 to 40kHz, and the driving voltage is 0 to 20V. One sub-beam 202 of the coupled fiber bundle 2 is wound around PZT923 and fixed. The driving of the PZT923 receives the instruction sent by the acquisition control unit 4, and the frequency and the voltage of the vibration of the PZT923 are controlled according to the requirement.

When the acquisition control unit 4 controls the PZT923 to vibrate, the mode disturbing effect is achieved on the light transmitted in the sub-beam 202, so that the speckle effect is inhibited, and the target area generates uniform light for illumination; when the acquisition control unit 4 controls the PZT923 to stop vibrating, the target region generates speckle illumination, and two illumination modes are generated accordingly, and the specific imaging method is the same as that in embodiment 6, and is not described again.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A fluorescent imaging illumination device based on the speckle principle is characterized by comprising a coupling optical fiber bundle and one or more mixed excitation light generating units;

the coupled fiber bundle comprises a plurality of sub-bundles;

2. The fluorescence imaging illumination device of claim 1, wherein the speckle particles of the speckle illumination satisfy the following relationship: the number of speckle particles in a single sampling window is 2-81; and satisfies the following conditions:

/Δs∈[i.4，9]

3. The fluorescence imaging illumination device of claim 1, wherein the numerical aperture of the fiber optic bundle is preferably ≥ 0.5; preferably, the coupling fiber bundle is composed of a plurality of multimode fibers, preferably plastic fibers.

4. The fluorescence imaging illumination device according to claim 1, wherein the mixed excitation light generating unit further comprises a mode filter for filtering the transmission mode of the light in the fiber bundle, and the mode filter filters out the low order mode and retains the high order mode.

5. The fluorescence imaging illumination device based on the speckle principle as claimed in claim 1, wherein the mixed excitation light generation unit comprises a coherent fluorescence excitation light source and an incoherent excitation light source, the coherent fluorescence excitation light source is preferably a laser, the incoherent excitation light source is preferably a high-power LED-narrowband filter combination, the coherent fluorescence excitation light source and the incoherent excitation light source are coupled to the illumination light path through different sub-beams of a coupling fiber bundle or coupled to the illumination light path through the same sub-beam after being combined by the beam combining device; the beam combining device is preferably an optical fiber beam combiner or a space beam combining device.

6. The fluorescence imaging illumination device based on the speckle principle as claimed in claim 1, wherein the mixed excitation light generation unit comprises a coherent fluorescence excitation light source and a speckle attenuation device, the coherent fluorescence excitation light source and the speckle attenuation device are connected in series and coupled into an illumination light path through a sub-beam, namely, the speckle attenuation device is arranged at the light exit, the conducting optical fiber and the rear end of the optical fiber; when the speckle attenuation device is started, the speckle effect is inhibited, and uniform light illumination spots are formed; when the speckle reduction device is turned off, speckle illumination is created.

7. An optical tomography image acquisition system based on the speckle principle, which is characterized by comprising the fluorescent imaging illumination device based on the speckle principle, a mirror body, an imaging device and an acquisition control device according to any one of claims 1 to 6;

8. The speckle-principle-based optical tomographic image acquisition system according to claim 7, wherein the mirror body comprises a dichroic mirror, an objective lens, a fluorescent filter, an imaging lens; a dichroic mirror and an objective lens are sequentially arranged on the illumination light path; the imaging light path is sequentially provided with an objective lens, a dichroic mirror, a fluorescent filter and an imaging lens; the microscope body is a micro fluorescent microscope, is worn on the body of an experimental animal during work and is fixed at a part needing imaging;

9. An imaging method using the optical tomographic image acquisition system based on the speckle principle as claimed in claim 7 or 8, comprising the steps of:

(1) controlling a fluorescence imaging lighting device of the system to alternately generate fluorescence excitation light for forming speckle illumination and fluorescence excitation light for forming uniform light illumination, and controlling the imaging device to perform speckle illumination imaging and uniform light illumination imaging to obtain an image sequence L of the same-size speckle illumination imaging_sAnd uniform light illumination imaging L_uThe image sequence of (1);

(2) according to the principle that the imaging phase difference time does not exceed a preset threshold value, illuminating each i of the image sequence imaged by the speckles with the same size obtained in the step (1)_sAnd each i of the image sequence imaged by the uniform light illumination_uPerforming correlation to obtain a speckle illumination imaging and uniform light illumination imaging correlation image pair sequence;

10. The imaging method according to claim 9, wherein the step (1) is a repetition of the following steps a1, a 2; or repeating the following steps B1 and B2;

A1. the acquisition control device sends a trigger signal to a coherent light source in the mixed excitation light generation unit to generate coherent fluorescence excitation light, and simultaneously or after waiting for delay, sends the trigger signal to an image acquisition device in the endoscope body, the image acquisition device starts to acquire signals, and the delay length is less than the difference between the duration time of the coherent fluorescence excitation light and the acquisition time of the image acquisition device; the coherent fluorescence exciting light is transmitted to the lens body through the coupling optical fiber bundle, speckle illumination is formed at the focal plane of the objective lens, and the image acquisition device acquires the speckle illumination at the momentA speckle-illuminated image i_s(ii) a After the acquisition is finished, the acquisition control device sends a trigger signal to close the coherent light source;

A2. the acquisition control device sends a trigger signal to an incoherent light source in the mixed excitation light generation unit to generate an incoherent fluorescence excitation light source, and simultaneously or after waiting for delay, sends a trigger signal to the image acquisition device, the image acquisition device starts to acquire signals, and the delay length is less than the difference between the duration time of the incoherent fluorescence excitation light and the acquisition time of the image acquisition device; the incoherent fluorescence excitation light is transmitted to the lens body through the coupling optical fiber bundle, uniform light illumination is formed at the focal plane of the lens body, and at the moment, the image acquisition device acquires an image i illuminated by the uniform light_u(ii) a After the acquisition is finished, the acquisition control device sends a trigger signal to close the incoherent light source;

B1. the acquisition control device sends a trigger signal to a coherent light source in the mixed excitation light generation unit to generate a coherent fluorescence excitation light source, and simultaneously or after waiting for delay, sends a trigger signal to an image acquisition device in the endoscope body, the image acquisition device starts to acquire signals, and the delay length is less than the difference between the duration time of the coherent fluorescence excitation light and the acquisition time of the image acquisition device. The light emitted by the light source is transmitted to the lens body through the coupling optical fiber bundle, the speckle illumination is formed at the focal plane of the objective lens, and at the moment, the image acquisition device acquires an image i of the speckle illumination_s；

B2. The collecting control device sends a trigger signal to the speckle attenuating device to enable the speckle attenuating device to start to operate, and simultaneously or after waiting for delay, sends the trigger signal to the image collecting device to enable the image collecting device to start to collect signals, wherein the delay length is smaller than the difference between the operating time of the speckle attenuating device and the collecting time of the image collecting device. At the moment, the light is transmitted to the lens body through the coupling optical fiber bundle and is homogenized by the speckle attenuation device, uniform light illumination is formed at the focal plane of the objective lens, and at the moment, the image acquisition device acquires an image i illuminated by the uniform light_u。