CN104677871A - Multi-photon exciting, illuminating and micro-imaging system of X-ray plate - Google Patents

Abstract

The invention provides a multi-photon exciting, illuminating and micro-imaging system of an X-ray plate. The multi-photon exciting, illuminating and micro-imaging system comprises a multi-photon laser unit, an illuminating unit and an imaging and detecting unit which are sequentially arranged on the system from front to back, wherein the multi-photo laser unit is used for generating and modulating laser and feeding the modulated laser into the illuminating unit; the illuminating unit is used for receiving the modulated laser to generate light sheet illumination and stimulate the to-be-detected sample to generate fluorescence; the imaging and detecting unit is used for detecting and converting fluorescence generated by the to-be-detected sample into a digital image. The multi-photon exciting, illuminating and micro-imaging system disclosed by the invention can be used for quickly imaging, so that living dynamic development imaging or nerve cell activation recording can be performed.

Description

Multiphoton Excitation Light Sheet Illumination Microscopic Imaging System

technical field

The invention belongs to the field of optics, in particular to fluorescence microscopic imaging technology.

Background technique

Existing fluorescence microscopic imaging can be divided into scanning microscopic imaging based on point-scanning imaging according to the imaging method, and light-sheet microscopic imaging using CCD to detect and image after irradiating fluorescence with a thin beam on the side; in addition, the existing Fluorescence microscopy imaging can be divided into single-photon excitation and two-photon excitation according to the fluorescence excitation conditions. Two-photon excitation is also called multi-photon excitation. Two-photon excitation refers to the fact that fluorescent molecules can absorb two long-wavelength Photons, after the excitation lifetime, emit photons with shorter wavelengths, so two-photon excitation can use light waves with longer wavelengths than single-photon excitation to excite fluorescence, that is, near-infrared light can be used for fluorescence excitation, and near-infrared light has two The first advantage is that the penetration depth is high, that is, the imaging depth is high, so it is suitable for observing thick samples, and the second is that the living samples to be detected have low toxicity, so they are suitable for observing biological living samples.

In the prior art, there is a multi-photon excitation scanning imaging technology that utilizes point-scanning imaging. Although this technology can detect living biological samples, the speed of using this technology to detect and generate images is slow because it obtains fluorescent images through point-by-point scanning. slow.

Contents of the invention

The invention provides a multi-photon excitation light sheet illumination microscopic imaging system with fast imaging speed, which is used to solve the technical defect of slow imaging speed in the existing microscopic imaging technology.

The present invention provides a microscopic imaging system for multi-photon excitation light sheet illumination, which includes: a multi-photon laser unit, an illumination unit, and an imaging detection unit located on the fluorescent light path of the imaging system from front to back;

The multiphoton laser unit is used to generate and modulate laser light and send the modulated laser light into the lighting unit; the lighting unit is arranged on the side of the sample to be detected, and is used to receive the modulated laser light and generate a The light sheet illuminates and excites the sample to be detected to generate fluorescence; the imaging detection unit is located above the sample to be detected, and is used to detect the fluorescence generated by the sample to be detected and convert it into a digital image, at least including a photoelectric device that detects the fluorescence and converts it into an electrical signal Probe camera.

In particular, the illumination unit includes a multiphoton light sheet generation optical path and an illumination objective lens positioned sequentially behind the fluorescence optical path of the imaging system;

The optical path generated by the multi-photon light sheet is used to receive the modulated laser light and generate a light sheet to be incident on the illumination objective lens; the illumination objective lens is used to receive the light sheet to illuminate again and excite the sample to be detected to generate fluorescence.

In particular, the light sheets received by the illumination objective lens are all perpendicular to the axis of the imaging detection unit, and the excitation position where the light sheets are irradiated on the sample to be detected is within the focal plane of the imaging detection unit.

In particular, the multi-photon laser unit includes a laser light source and a multi-photon excitation optical path positioned sequentially behind the fluorescence optical path of the imaging system;

The laser light source is used to generate laser light with high photon density and send it into the multi-photon excitation optical path, and the multi-photon excitation optical path is used to receive and modulate the laser light and send it into the illumination unit.

In particular, the imaging detection unit includes an object stage, an imaging objective lens, and a filter that are sequentially positioned behind the fluorescence optical path of the imaging system;

The stage is used to fix and change the excitation position of the sample to be detected, so that the sample to be detected is excited to generate fluorescence at multiple angles or depths; the imaging objective lens is located above the sample to be detected, and is used to focus the fluorescence and sent to the filter; the filter is fixedly arranged in front of the optical path of the photodetection camera, and is used to filter out the stray light in the fluorescence.

In particular, the stage includes at least a micro-operation controller, and the micro-operation controller is used to perform translation or rotation around the vertical axis of the sample to be detected, so as to adjust the relationship between the sample to be detected and the optical path of the imaging system. relative position.

In particular, the imaging detection unit also includes an electrical signal acquisition control module and a central processing module, the electrical signal acquisition control module is specifically used to receive the electrical signal output by the photoelectric detection camera and convert it into a digital signal, and the central processing The module is specifically used for receiving said digital signal and generating a digital image.

The beneficial effects of the present invention are:

The multiphoton excitation light sheet illumination microscopic imaging system of the present invention detects and images the fluorescence generated by the multiphoton excitation of the sample to be detected by using the light sheet illumination microscopic imaging technology, so that the corresponding imaging detection unit can perform parallel image acquisition, thereby compared The multiphoton laser point scanning microscopy imaging in the prior art improves the imaging speed, while the multiphoton excitation improves the detection depth due to the use of near-infrared excitation light, that is to say, it can achieve a higher imaging speed under the condition of ensuring the imaging speed. Detection depth, so it can realize dynamic imaging of thicker living organisms; due to the combination of the imaging speed of light sheet illumination micro-imaging that can realize parallel image acquisition and the imaging depth of two-photon excitation, it is in medical imaging. field, the present invention can be used for dynamic developmental imaging and neuronal activity recording of living organisms such as zebrafish and fruit flies.

Description of drawings

Fig. 1 is the block diagram of the structure of a multiphoton excitation light sheet illumination microscopic imaging system according to an embodiment of the present invention;

Fig. 2 is a structure diagram of a multi-photon excitation light sheet illumination microscopic imaging system according to an embodiment of the present invention;

In the figure: 1-multiphoton laser unit, 11-laser light source, 12-multiphoton excitation optical path, 2-illumination unit, 21-multiphoton light sheet generation optical path, 22-illumination objective lens, 3-imaging detection unit, 31-carrier Object stage, 32-imaging objective lens, 33-filter, 34-photoelectric detection camera, 35-electrical signal acquisition control module, 36-central processing module.

Detailed ways

Fig. 1 is a structural block diagram of a multi-photon excitation light sheet illumination micro-imaging system according to an embodiment of the present invention, and Fig. 2 is a structure diagram of a multi-photon excitation light sheet illumination micro-imaging system according to an embodiment of the present invention, as shown in Fig. 1 and Fig. 2 As shown, the multiphoton excitation light sheet illumination microscopic imaging system of the present invention includes: a multiphoton laser unit 1, an illumination unit 2, and an imaging detection unit 3 located on the fluorescence optical path of the imaging system from front to back;

The multiphoton laser unit 1 is used to generate and modulate laser light and send the modulated laser light into the illumination unit; the illumination unit 2 is arranged on the side of the sample to be detected, and is used to receive the modulated laser light and then Generate a light sheet to illuminate and excite the sample to be detected to generate fluorescence; the imaging detection unit 3 is located above the sample to be detected, and is used to detect the fluorescence generated by the sample to be detected and convert it into a digital image, at least including detecting the fluorescence and converting it into an electric Signal photoelectric detection camera 34; preferably, the photoelectric detection camera can be a high-speed SCOMS chip camera, which can quickly detect the fluorescence produced by the sample to be detected.

Preferably, the illumination unit 2 includes a multiphoton light sheet generating optical path 21 and an illuminating objective lens 22 positioned sequentially behind on the fluorescence optical path of the imaging system;

The multi-photon light sheet generation optical path 21 is used to receive the modulated laser and generate a light sheet to be incident on the illumination objective lens 22; the illumination objective lens 22 is used to receive the light sheet to illuminate and excite the sample to be detected Produce fluorescence.

Preferably, the light sheet received by the illumination objective lens 22 is perpendicular to the axis of the imaging detection unit 3, that is, the axis of the imaging objective lens 32, and the excitation position where the light sheet is irradiated on the sample to be detected is within the range of the imaging detection unit. The focal plane of the unit 3 is also the focal plane of the imaging objective lens 32 .

Preferably, the multiphoton laser unit 1 includes a laser light source 11 and a multiphoton excitation optical path 12 positioned sequentially behind the fluorescence optical path of the imaging system;

The laser light source 11 is used to generate high-photon density laser light and send it into the multi-photon excitation optical path 12, and the multi-photon excitation optical path 12 is used to receive and modulate the laser light and send it into the illumination unit 2; preferably , the laser light source 11 is an ultrafast femtosecond pulse laser, which can generate excitation light with high photon density; the multiphoton excitation optical path 12 includes optical elements such as a laser beam expander, a mirror, and a scanning galvanometer, which are specifically used for The received laser light generated by the laser light source is modulated and sent to the multi-photon optical sheet generating optical path 21 in the illumination unit 2 .

Preferably, the imaging detection unit 3 includes an object stage 31, an imaging objective lens 32, and a filter 33 that are sequentially positioned behind on the fluorescence optical path of the imaging system;

The stage 31 is used to fix and change the excitation position of the sample to be detected, so that the sample to be detected is excited to generate fluorescence at multiple angles or depths to generate a single slice image; the imaging objective lens 32 is positioned at the sample to be detected The upper part is used to focus the fluorescence and send it into the filter 33; the filter 33 is fixedly arranged in front of the light path of the photodetection camera 34, and is used to filter out the stray light in the fluorescence.

Preferably, the stage 31 includes at least a micro-operation controller, and the micro-operation controller is used to perform translation or rotation around the vertical axis of the sample to be detected, so as to adjust the optical path between the sample to be detected and the imaging system The relative position of the sample to be detected can move in three-dimensional space and rotate in the vertical direction, so that the illumination unit 2, that is, the illumination objective lens 22, can excite the sample to be detected to generate fluorescence from multiple angles and layers, so as to generate Multiple slice images of the sample, and then perform three-dimensional reconstruction on the slice images to obtain a three-dimensional image of the sample to be detected.

Preferably, the imaging detection unit 3 further includes an electrical signal acquisition control module 35 and a central processing module 36, the electrical signal acquisition control module 35 is specifically configured to receive the electrical signal output by the photoelectric detection camera and convert it into a digital signal, The central processing module 36 is specifically used to receive the digital signal and generate a digital image, that is, the central processing module 36 is specifically used to receive the electrical signal converted and output by the photodetection camera 34 according to the detected fluorescence. And it is stored and processed, so as to finally obtain the high-resolution digital image of the sample to be detected; preferably, the central processing module 36 can be a computer, and the central processing module 36 is equipped with a graphics processing card slot and can Support graphics parallel computing, which is used to continuously convert the collected electrical signal corresponding to the fluorescence emitted by the sample to be detected into a digital image. At this time, the digital image is a two-dimensional slice image of the sample to be detected, and then the collected digital image Perform position registration, image fusion, image reconstruction and other processing, and finally perform three-dimensional reconstruction on the image to obtain a three-dimensional image of the sample to be detected.

Preferably, the electrical signal acquisition control module 35 is also respectively connected to and inputs the control signal sent by the central processing module 36 to the micro-operation controller of the laser light source 11 and the stage 31; preferably, to realize the central The processing module takes the electrical signal acquisition control module of the stage as an example, the communication control interface for connecting the stage is installed on the central processing module 36, this interface can be a USB interface or a serial interface, and these interfaces pass through the USB line Or the serial line is connected to the stage, and parameters such as its position and speed can be read from the stage 31, and the obtained parameters are returned to the central processing module 36; that is, the central processing module 36 is specifically used to pass The electrical signal acquisition control module sends control instructions to the micro-operation controller of the stage 31 to control the three-dimensional movement and rotation of the sample to be detected, and receives the motion parameters of the micro-operation controller of the stage 31; the central processing module 36 The control of the laser light source 11 is similar to this and will not be repeated here.

The multiphoton excitation light sheet illumination microscopic imaging system of the present invention detects and images the fluorescence generated by the multiphoton excitation of the sample to be detected by using the light sheet illumination microscopic imaging technology, so that the corresponding imaging detection unit can perform parallel image acquisition, thereby compared The multiphoton laser point scanning microscopy imaging in the prior art improves the imaging speed, while the multiphoton excitation improves the detection depth due to the use of near-infrared excitation light, that is to say, it can achieve a higher imaging speed under the condition of ensuring the imaging speed. Detection depth, so it can realize dynamic imaging of thicker living organisms; due to the combination of the imaging speed of light sheet illumination micro-imaging that can realize parallel image acquisition and the imaging depth of two-photon excitation, it is in medical imaging. field, the present invention can be used for dynamic developmental imaging and neuronal activity recording of living organisms such as zebrafish and fruit flies.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. scope.

Claims (7)

1. multiphoton excitation mating plate illumination a micro imaging system, it is characterized in that, comprise be positioned at described imaging system from front to back successively fluorescence light path on: multi-photon laser cell, lighting unit, image checking unit;

Described multi-photon laser cell, for generation of with modulated laser and by modulation after laser send into lighting unit; Described lighting unit is located at the side of described sample to be detected, produces a mating plate again throw light on and excite sample to be detected to produce fluorescence for the laser after receiving described modulation; Described image checking unit is positioned at above sample to be detected, for detecting the fluorescence of sample to be detected generation and transferring digital picture to, at least comprises and detects described fluorescence and the photodetection camera transferring electric signal to.

2. multiphoton excitation mating plate according to claim 1 illumination micro imaging system, is characterized in that, on the fluorescence light path that described lighting unit is included in described imaging system position successively multi-photon mating plate rearward produce light path and an illumination objective lens;

Described multi-photon mating plate produces light path and produces a mating plate again thus incident described illumination objective lens for the laser after receiving described modulation; Described illumination objective lens is for receiving described mating plate thus throwing light on and excite sample to be detected to produce fluorescence.

3. multiphoton excitation mating plate illumination micro imaging system according to claim 2, it is characterized in that, the mating plate that described illumination objective lens receives and described mating plate vertical with the axle of described image checking unit impinges upon and sample to be detected excites position in the focal plane of described image checking unit.

4. multiphoton excitation mating plate according to claim 1 illumination micro imaging system, is characterized in that, position LASER Light Source rearward and multiphoton excitation light path successively on the fluorescence light path that described multi-photon laser cell is included in described imaging system;

Described LASER Light Source for generation of high photon density laser and send into described multiphoton excitation light path, described multiphoton excitation light path be used for receive and modulate described laser and send into described lighting unit.

5. multiphoton excitation mating plate according to claim 1 illumination micro imaging system, is characterized in that, position objective table, image-forming objective lens and filter plate rearward successively on the fluorescence light path that described image checking unit is included in described imaging system;

Described objective table be used for fixing and change sample to be detected excite position, be excited the fluorescence produced in multiple angle or the degree of depth to make sample to be detected; Described image-forming objective lens is positioned at above sample to be detected, for focusing on described fluorescence and sending into described filter plate; Described filter plate is fixedly installed on the described light path front of described photodetection camera, for the veiling glare in fluorescence described in filtering.

6. multiphoton excitation mating plate illumination micro imaging system according to claim 5, it is characterized in that, described objective table at least comprises microoperation controller, described microoperation controller is used for the operation that sample to be detected is performed to translation or rotates around vertical axes, to adjust the relative position of described sample to be detected and described imaging system light path.

7. multiphoton excitation mating plate illumination micro imaging system according to claim 1, it is characterized in that, described image checking unit also comprises electrical signal collection control module and central processing module, described electrical signal collection control module is specifically for receiving the electric signal of described photodetection camera output and being converted to digital signal, and described central processing module is specifically for receiving described digital signal and generating digital picture.