WO2016061710A1

WO2016061710A1 - Fast wide field-of-view volume holographic fluorescence micro-imaging system

Info

Publication number: WO2016061710A1
Application number: PCT/CN2014/000935
Authority: WO
Inventors: 罗建文; 吕衍路; 白净; 刘飞
Original assignee: 清华大学
Priority date: 2014-10-21
Filing date: 2014-10-21
Publication date: 2016-04-28

Abstract

A fast wide field-of-view volume holographic fluorescence micro-imaging system, including a laser light source (1), a dichroic spectroscope (2), a micro-objective (3), an MEMS micro-mirror array device (4), a volume holographic grating device (5), an imaging lens (6) and an image detector array (7). The laser light source (1) provides intensity-uniform illumination conditions for an imaging target (8) and emits a lighting optical wave to the dichroic spectroscope (2). The dichroic spectroscope (2) emits the lighting optical wave to the imaging target (8). The fluorescent light emitted by the imaging target (8) returns to the dichroic spectroscope (2). The dichroic spectroscope (2) filters the lighting optical wave and projects the fluorescent light to the MEMS micro-mirror array device (4) via the micro-objective (3). The MEMS micro-mirror array device (4) carries out angular coding on light waves at different positions according to a central wavelength of an imaging spectrum and a Bragg characteristic parameter of the volume holographic grating device (5) to further control a deflection direction of an imaging light beam, and the deflected imaging light beam is incident on the volume holographic grating device (5) at a matching angle. The volume holographic grating device (5) diffracts the incident light subjected to spatial angular coding, and diffraction light is imaged to the image detector array (7) via the imaging lens (6).

Description

Fast wide field holographic fluorescence microscopy imaging system

Technical field

The present invention relates to the field of spatial spectral microscopic imaging technology, and more particularly to a fast wide field volume holographic fluorescence microscopy imaging system.

Background technique

Spectral microscopy imaging system based on volume holographic grating is a new type of microscopic imaging technology that has attracted much attention in recent years. It has a very broad field in shallow tumor detection, disease diagnosis, food and drug supervision, biological inspection and quarantine, material science and other fields. Application prospects. The volume holographic microscopy imaging system utilizes the Bragg selection characteristics of a volume holographic grating device to achieve microscopic imaging with high spectral resolution. The volume holographic grating device is formed by recording a coherent light of two beams of coherent light on a surface of a recording material by using a photorefractive material having a certain thickness. The recorded hologram pattern is a three-dimensional phase grating having a thickness, and the three-dimensional phase grating It has special diffraction characteristics, fine spectral angle selection characteristics, volume multiplexing characteristics, and a very wide spectral adjustment range, enabling continuous adjustment over a wide spectral range. When a certain wavelength of light is used to illuminate the biological tissue, the surface of the biological tissue will scatter and absorb the incident light. When the scattered light passes through the microscope objective, it is incident on the surface of the volume holographic grating located at the focal plane of the image, and the volume holographic grating The Bragg selection characteristic allows only incident light waves that match the angle of incidence to pass through with higher diffraction efficiency, thereby obtaining an image of the narrow-band spectrum of the tissue and avoiding interference from the background light.

The volume holographic optical imaging system can realize the imaging of the narrow field of view under the narrow-band light source and obtain the spectral information of the corresponding region. The volume multiplexed volume holographic grating can acquire the three-dimensional tomographic image of the translucent tissue and the contour imaging of the non-transparent structure. Due to the degeneracy of the volume holographic grating, when the surface of the object is reflected or emitted with a broad spectrum of light, the image appearing on the back focal plane of the imaging lens is an image containing multiple color lights. If an image of a single color light is desired, filtering is required. The light is filtered, or the target is illuminated by using a planar beam splitting device, but both of these schemes increase the complexity of the system, and cannot achieve continuous fast switching of a single spectrum, thereby reducing imaging efficiency.

Summary of the invention

In view of the above problems, it is an object of the present invention to provide a fast wide field volume holographic fluorescence microscopy imaging system that can be used to image a broad spectrum imaging target and that can achieve fast switching of multiple spectra.

In order to achieve the above object, the present invention adopts the following technical solution: a fast wide field volume holographic fluorescence microscopy imaging system, which comprises a laser light source, a dichroic beam splitter, a microscope objective, and a MEMS. a micromirror array device, an integrated holographic grating device, an imaging lens, and an image detector array, wherein the MEMS micromirror array device is located in front of a rear focal plane of the microscope objective, and the volume holographic grating device is located at Where the image focal plane of the microscope objective coincides with the focal plane of the imaging lens; the laser source is used to provide uniform illumination conditions for an imaging target, the laser source emitting illumination light to the a dichroic beam splitter; the dichroic beam splitter converts the illumination light wave to illuminate the imaging target perpendicular to its incident direction, and the fluorescent emission emitted by the imaging target returns to the dichroic beam splitter, the two directions a color splitter filters the illumination light wave and projects the fluorescence emitted by the imaging target onto the MEMS micromirror array device through the microscope objective; the MEMS micromirror array device according to a central wavelength of the imaging spectrum and the The Bragg characteristic parameters of the volume holographic grating device angularly encode the light waves at different positions to control the deflection direction of the imaging beam, and are deflected. The imaging beam is incident at an angle to match the volume holographic grating device; said volume holographic grating device through the incident light diffracted encoding spatial angle, diffracted light imaged through the imaging lens to the image detector array.

The volume holographic grating device adopts a single-path reflective volume holographic grating device, a single-channel transmissive volume holographic grating device, a multi-channel wavelength multiplexing reflective volume holographic grating device or a multi-channel wavelength multiplexing transmissive volume holographic grating device.

The spatial angle coding manner of the MEMS micromirror array device according to the imaging target feature and the imaging spectral requirement specifically includes the following two forms: 1 when the single spectral image information of the imaging target needs to be acquired, according to the volume holographic grating Matching conditions and imaging wavelengths control the deflection angle of each column of micromirrors of the MEMS micromirror array device, enabling imaging light waves to be incident on the volume holographic grating device at a Bragg-matched angle; 2 when continuous acquisition of the imaging is required Controlling each column of micromirrors of the MEMS micromirror array device according to matching conditions of the volume holographic grating device and wavelength parameters of different imaging spectra selected when a plurality of different wavelength images of the target continuously change in a short time The deflection angle is achieved by encoding the deflection angle of the MEMS micromirror array device to achieve fast switching of the imaging spectrum.

The invention adopts the above technical solutions, and has the following advantages: 1. The invention adopts the MEMS micro-mirror array device to spatially encode the imaging region, and combines the selection characteristics of the volume holographic grating device to effectively improve the volume holographic multi-spectral fluorescence microscopy. The imaging field of view width of the system can significantly improve the spectral switching speed, and can realize single-spectral imaging and multi-spectral imaging according to the characteristics of the imaging target and the requirements of the imaging spectrum. Compared with the existing spectral imaging system, It is easy to realize wide-field single-spectrum imaging, and it can also achieve continuous fast switching of single spectrum, ie multi-spectral imaging. 2. When the surface of the imaging target reflects or emits a broad spectrum of light, the image appearing on the back focal plane of the imaging lens is an image containing multiple color lights. The present invention rapidly adjusts and switches the MEMS micromirror array device by spatial angle coding. The angle of inclination so that the corresponding wavelength in the field of view satisfies the Bragg condition of the incident holographic grating, without the need for filter filtering Directly obtaining an image of a single color light in a wider field of view of the imaging target effectively improves the speed of imaging between different spectra, and achieves multiple continuous spectral imaging of a wide field of view. 3. The invention can obtain a plurality of continuous spectral images of an imaging target without using a light splitting device when imaging an imaging target that emits fluorescence, and can quickly switch MEMS when real-time observation of different spectral images of living tissue at different times is needed. The tilt angle of each column of micromirrors of the micromirror array device changes the spatial angle encoding of the reflected light to quickly realize multiple continuous spectral imaging. The invention has the advantages of simple structure, convenient use and high imaging efficiency, and can be widely applied to fluorescence microscopic imaging of transparent or translucent imaging targets.

DRAWINGS

The invention is described in detail below with reference to the accompanying drawings. It is to be understood, however, that the appended claims

1 is a schematic structural view of a volume holographic fluorescence microscopic imaging system of the present invention, wherein the volume holographic grating device of (a) is transmissive, and the volume holographic grating device of (b) is of a reflective type;

2 is a schematic diagram of the multi-wavelength working principle of the transmissive volume holographic grating of the present invention, wherein (a) is a schematic diagram of a single-wavelength working principle of a transmissive volume holographic grating, and (b) a transmissive volume holographic grating multi-wavelength recorded by wavelength multiplexing. Schematic diagram of the working principle, (c) is a schematic diagram of a multi-spectral imaging optical path of a wavelength-multiplexed transmissive volume holographic grating;

3 is a schematic diagram of the multi-wavelength working principle of the reflective volume holographic grating of the present invention, wherein (a) is a schematic diagram of a single-wavelength working principle of a reflective volume holographic grating, and (b) a reflective volume holographic grating recorded by wavelength multiplexing. Schematic diagram of the working principle of the wavelength, (c) is a schematic diagram of the wavelength multiplexed reflective holographic multi-spectral imaging optical path principle;

4 is a schematic structural view of a MEMS micromirror array device of the present invention, wherein (a) is a schematic structural view of a MEMS micromirror array device, and (b) is a structural principle of a micromirror in a MEMS micromirror array device. Schematic, (c) is a schematic diagram of the working principle of the MEMS micromirror device;

5 is a schematic diagram of the principle of single-spectrum spatial angle coding according to the present invention, wherein (a) is a schematic diagram of a single-spectrum spatial angle coding principle, and (b) is a surface light pattern of a volume holographic grating surface during single-spectrum imaging;

6 is a schematic diagram of the multi-spectral spatial angle coding principle of the present invention, wherein (a) is a schematic diagram of a multi-spectral switching spatial angle encoding principle, and (b) is an incident light pattern of color light of different wavelengths on a volume holographic grating grating.

detailed description

As shown in FIG. 1, the fast wide field volume holographic fluorescence microscopy imaging system of the present invention comprises a laser light source 1, a dichroic beam splitter 2, a microscope objective lens 3, a MEMS micro mirror array device 4, and an integrated body. Holographic grating a lens 5, an imaging lens 6 and an image detector array 7, wherein the MEMS micromirror array device 4 is located in front of the back focal plane of the microscope objective 3, and the volume holographic grating device 5 is located in the image focal plane of the microscope objective 3 The imaging lens 6 is at a position where the object focal planes coincide.

The laser light source 1 is used to provide the imaging target 8 with uniform intensity illumination conditions, the laser light source emits illumination light waves to the dichroic beam splitter 2; the dichroic beam splitter 2 converts the illumination light wave into the imaging target 8 perpendicular to its incident direction, The fluorescence emission from the imaging target 8 is returned to the dichroic beam splitter 2, which is used to filter out the illumination light wave and project the fluorescence emitted from the imaging target 8 through the microscope objective 3 to the MEMS for image spatial angle coding. The micromirror array device 4; the MEMS micromirror array device 4 calculates the reflection angle of the center wavelength of the imaging spectrum matched with the volume holographic grating device 5 according to the Bragg condition, and obtains the spatial angle coding of each column of the micro mirror, and according to the spatial angle The encoding controls the driving force of each column of micromirrors, thereby controlling the deflection directions of the columns of micromirrors, so that the deflected imaging beam is incident on the volume holographic grating device 5 at an angle satisfying the Bragg condition; the volume holographic grating device 5 passes through the space The angle-encoded incident light is diffracted and transmitted to the imaging lens 6; the imaging lens 6 is a volume holographic grating device The diffracted light is imaged to the image detector array 7.

In a preferred embodiment, as shown in FIGS. 2 and 3, the volume hologram grating device 5 may be a single-channel or wavelength-multiplexed reflective volume holographic grating device or a transmissive bulk holographic grating device.

In a preferred embodiment, as shown in FIG. 4(a), the MEMS micromirror array device 4 is an array of tens of thousands of micromirrors arranged neatly, and each of the micromirrors may have a rectangular or diamond shape. The MEMS micromirror array device comprises a micro mirror array, a micromirror array driving circuit and a corresponding micro controller, the micro controller is used for controlling the spatial angle encoding state of the micro mirror array, and the driving circuit is used for controlling the micro mirror array. The switch and the micromirror array operate. As shown in FIG. 4(b), each MEMS micromirror includes a micro mirror surface 41, a driving mechanism 42 and a supporting substrate 43. The micro mirror surface 41 is connected to the supporting substrate 43 through a driving mechanism 42. 41 can be tilted at any angle in a two-dimensional plane under the movement of the bottom drive mechanism 42 to control the direction of reflection of light incident on the mirror surface. As shown in FIG. 4(c), the basic principle of the MEMS micromirror array device is that the microcontroller sets the deflection parameters of each column of micromirrors according to the imaging spectral parameters and the volume holographic grating device parameters and sends them to the driving. a circuit, the driving circuit controls the driving mechanism according to the deflection angle of each column of micromirrors for the deflection angle of each micromirror, that is, by addressing the landing electrode of the associated M1 _{, i} column micromirror, the driving mechanism The free end is tilted to one side of the landing electrodes on both sides, and the tilt angle is ±12°, thereby adjusting the tilt angle of the micro-mirror surface. Among them, the MEMS micromirror array device can adopt a deformable mirror array as needed.

In a preferred embodiment, the imaging target 8 may employ a transparent or opaque biological tissue disposed at the anterior focal plane of the microscope objective 3.

In a preferred embodiment, the laser source 1 can employ a planar laser source.

The fast volume holographic fluorescence microscopy imaging system of the present invention can perform single-spectrum imaging or fast multi-spectral imaging according to the characteristics of the imaging target and the requirements of the imaging spectrum. The following two cases are described in detail through specific embodiments:

Embodiment 1: The single-spectral imaging is performed by using the fast wide-field volume holographic fluorescence microscopic imaging system of the present invention, that is, the single-spectral image information of the imaging target is acquired.

When it is required to acquire single-spectral image information of the imaging target, the deflection angle of each column of the micro-mirrors of the MEMS micromirror array device 4 is set according to the matching condition of the volume hologram grating device 5 and the imaging spectral wavelength, so that the imaging light wave can be matched by Bragg The angle is incident on the corresponding region of the volume hologram grating device 5.

As shown in FIG. 1(a), the laser light source 1 emits an illumination light wave of uniform intensity transmitted to the imaging target 8 via the dichroic beam splitter 2, and the dichroic beam splitter 2 filters out the illumination light wave and emits the fluorescent light emitted by the imaging target 8. The microscope objective 3 is projected onto the MEMS micromirror array device 4, and the MEMS micromirror array device 4 designs a spatial encoding angle according to the center wavelength of the imaging band and the parameters of the volume hologram grating device, and controls the deflection direction of the imaging beam, after being deflected The imaging beam is incident on the transmissive volume hologram grating device 5 at a matching angle and finally imaged to the image detector array 7 via the imaging lens 6.

The single-spectral spatial angle coding principle is as follows: as shown in FIG. 5, the light wave emitted by the imaging target 8 is incident on the MEMS micro-mirror array device 4 through the microscope objective lens 3, and the tilted micro-mirror shown in FIG. 5(a) Representing a row of micromirrors, Figure 5(b) shows the incident light pattern on the surface of a volume holographic grating during single-spectral imaging, and micro-reflection with the same spatial angle encoding when calculating the spatial angle encoding according to the degenerate characteristics of the volume holographic grating device The number of columns n, n of the mirror can be calculated by the following formula

Where l is the incident width of the microscope objective on the MEMS micromirror array device and d is the diameter of the single micromirror.

The object light wave is reflected by the MEMS micromirror array device 4 to the corresponding region of the volume holographic grating device 5, and the incident light wave has the same wavelength, and the light wave of different regions in the negative x direction adopts the same spatial angle encoding method on the volume holographic grating device 5. It is to evenly arrange image strips of approximately equal width, and the stripe width can be calculated by the following formula.

l _i =n _i d/cos2θ _i ≈n _i d/2θ _i

Where l _i is the width of the ith image stripe, d is the diameter of a single micromirror, θ _i is the spatial encoding angle, and n _i is the column of micromirrors with the same deflection angle on the MEMS micromirror array device number.

Embodiment 2: The fast multi-spectral imaging is completed by using the fast volume holographic fluorescence microscopic imaging system of the present invention, that is, a plurality of different spectral images in which the imaging target continuously changes in a short time are continuously acquired.

When it is required to continuously acquire a plurality of different spectral images in which the imaging target continuously changes in a short time, the longitudinal dimensions of the MEMS micromirror array device are designed according to the matching conditions of the volume holographic grating device and the wavelength parameters of the selected different imaging spectra. The deflection angle of the mirror and the angular switching speed and range (this optical device has been eliminated) enable fast imaging spectral switching.

As shown in FIG. 1(b), the laser light source 1 emits an illumination light having uniform intensity and is emitted to the imaging target 8 via the dichroic beam splitter 2, and the dichroic beam splitter 2 filters out the illumination light wave and emits the fluorescent light emitted by the imaging target 8. The microscope objective 3 is projected onto the MEMS micromirror array device 4, and the MEMS micromirror device array 4 performs image space angle coding, and designs a spatial coding angle according to different imaging spectral wavelength parameters and parameters of the volume holographic grating device, and controls the MEMS micro. The tilting angle and the angular switching speed of the corresponding micro-mirrors on the mirror array device, the deflected imaging beam is incident on the reflective volume hologram grating device 5 at a matching angle, and finally the imaging lens 6 diffracts the volume holographic grating device 5. Light is imaged into the image detector array 7.

As shown in FIG. 4, the multi-spectral spatial angle coding principle is that the light wave emitted from the imaging target 8 passes through the microscope objective 3 and is incident on the MEMS micromirror array device 4, as shown in FIG. 6(a). The mirrors represent micromirrors that correspond to the same column. The object light waves are projected through the MEMS micro-mirror array device array 4 to corresponding regions of the volume holographic grating device 5, and the wavelengths of the incident light waves are different, and the different wavelengths of the light waves in the x-negative direction are respectively switched to θ ₁ , θ ₂ by different tilt angles. ,..., the spatial coding angle of θ _n . As shown in FIG. 6(b), the incident position of the color light of different wavelengths on the volume hologram grating device 5 also moves as the deflection angle of the micro mirror array changes, wherein the stripe width can be calculated by the following formula:

l _i =n _i d/cos2θ _i ≈n _i d/2θ _i

Where l _i is the corresponding image stripe width, d is the diameter of a single micromirror, θ _i is the spatial encoding angle, and n _i is the number of columns of micromirrors having the same deflection angle on the MEMS micromirror array device.

The above embodiments are only used to illustrate the present invention, wherein the structure, arrangement position, connection mode, structural parameters of the device, coding mode, and data processing method of each component may be changed, which is the basis of the technical solution of the present invention. Equivalent transformations and improvements made above should not be excluded from the scope of the present invention.

Claims

A fast wide field volume holographic fluorescence microscopy imaging system, comprising: a laser light source, a dichroic beam splitter, a microscope objective, a MEMS micro mirror array device, an integrated holographic grating device, and a An imaging lens and an image detector array, wherein the MEMS micromirror array device is located in front of a focal plane of the microscope objective, the volume holographic grating device is located at an image focal plane of the microscope objective and At a position where the focal planes of the imaging lens coincide;

The laser light source is configured to provide uniform illumination conditions for an imaging target, the laser light source emitting illumination light waves to the dichroic beam splitter; the dichroic beam splitter transforms illumination light waves perpendicular to an incident direction thereof Irradiating the imaging target, the fluorescent emission emitted by the imaging target is returned to the dichroic beam splitter, and the dichroic beam splitter filters out the illumination light wave and projects the fluorescence emitted by the imaging target through the microscope objective lens To the MEMS micromirror array device; the MEMS micromirror array device angularly encodes light waves at different positions according to a central wavelength of the imaging spectrum and a Bragg characteristic parameter of the volume holographic grating device to control deflection of the imaging beam a direction, the deflected imaging beam is incident on the volume holographic grating device at a matching angle; the volume holographic grating device diffracts the spatially angled incident light, and the diffracted light is imaged to the image by the imaging lens Detector array.
A fast wide field volume holographic fluorescence microscopy imaging system according to claim 1, wherein said volume holographic grating device comprises a single-channel reflective volume holographic grating device, a single-channel transmissive volume holographic grating device, A multiplexed wavelength multiplexed reflective volume holographic grating device or a multiplexed wavelength multiplexed transmissive volume holographic grating device.
A fast wide field volume holographic fluorescence microscopy imaging system according to claim 1 or 2, wherein the spatial angle coding mode of the MEMS micromirror array device is based on imaging target characteristics and imaging spectral requirements, It includes the following two forms:

1 when it is required to acquire single-spectral image information of the imaging target, controlling a deflection angle of each column of the micro-mirror of the MEMS micro-mirror array device according to a matching condition and an imaging wavelength of the volume holographic grating, so that the imaging light wave can be a Bragg matching angle is incident on the volume holographic grating device;

2 when it is required to continuously acquire a plurality of different wavelength images of the imaging target that continuously change in a short time, controlling the MEMS micro-reflection according to matching conditions of the volume holographic grating device and wavelength parameters of different imaging spectra selected The deflection angle of each column of micromirrors of the mirror array device enables fast switching of the imaging spectrum by encoding the deflection angle of the MEMS micromirror array device.