阅读说明：本技术 一种快速光谱显微成像装置 (Quick spectral microscopic imaging device ) 是由 颜成钢 吕彬彬 孙垚棋 张继勇 李宗鹏 张勇东 于 2021-03-31 设计创作，主要内容包括：本发明公开了一种快速光谱显微成像装置,包括依次设置的宽光谱白光光源、载物台、显微物镜、分光棱镜；沿分光棱镜反射光方向设置有第一视场光阑、第一4F中继透镜和第一灰度相机；沿分光棱镜出射光方向设置的第二视场光阑、第二4F中继透镜、闪耀光栅、带通滤波器、第三4F中继透镜、微透镜阵列、第四4F中继透镜和第二灰度相机。第二4F中继透镜的两组镜片之间设置有孔径光阑。本发明可以实现对三维光谱图像的单次曝光获取,并且两个通道分别获取高空间分辨率图像和低分辨率光谱图像,弥补像素分区复用引起的空间分辨率损失；发明装置可以实时获取场景光谱数据并且实时显示,数据获取和数据处理过程没有时间延迟。(The invention discloses a rapid spectral microimaging device, which comprises a broad spectrum white light source, an objective table, a microobjective and a beam splitter prism which are arranged in sequence; a first field diaphragm, a first 4F relay lens and a first gray camera are arranged along the direction of the light reflected by the beam splitter prism; and the second field diaphragm, the second 4F relay lens, the blazed grating, the band-pass filter, the third 4F relay lens, the micro-lens array, the fourth 4F relay lens and the second gray camera are arranged along the emergent light direction of the beam splitter prism. An aperture diaphragm is arranged between the two groups of lenses of the second 4F relay lens. The invention can realize the single exposure acquisition of the three-dimensional spectral image, and the two channels respectively acquire the high-spatial resolution image and the low-resolution spectral image, thereby making up the spatial resolution loss caused by pixel partition multiplexing; the device can acquire scene spectrum data in real time and display the scene spectrum data in real time, and the data acquisition and data processing processes have no time delay.)

1. A rapid spectral microimaging device is characterized by comprising a broad spectrum white light source (1), an objective table (2), a microobjective (3) and a beam splitter prism (4) which are arranged in sequence; a first field diaphragm (5.1), a first 4F relay lens (7.1) and a first gray camera (11.1) are arranged along the direction of the reflected light of the beam splitter prism (4); a second field diaphragm (5.2), a second 4F relay lens (7.2), a blazed grating (8), a band-pass filter (9), a third 4F relay lens (7.3), a micro-lens array (10), a fourth 4F relay lens (7.4) and a second gray camera (11.2) are arranged along the emergent light direction of the beam splitter prism (4);

an aperture diaphragm (6) is arranged between the two groups of lenses of the second 4F relay lens (7.2);

the broad spectrum white light source (1) is used for illuminating an observation object, namely a sample, on the objective table (2), the microscope objective (3) is used for acquiring two-dimensional image information of the sample on the objective table (2) and dividing the two-dimensional image information into two beams of emergent light and reflected light after passing through the beam splitter prism (4):

the reflected light is imaged on a plane where a first field diaphragm (5.1) is located at a brightness of a, and a real image of the first field diaphragm surface is relayed to the plane where a first gray-scale camera (11.1) is located through a first 4F relay lens (7.1);

emergent light is focused on a plane where a second field diaphragm (5.2) is located at the brightness of 1-a, a real image of the second field diaphragm surface is relayed to the surface of a blazed grating (8) through a second 4F relay lens (7.2), and an aperture diaphragm (6) is used for controlling the numerical aperture of incident light; the blazed grating (8) disperses the transmitted light of different spectral wavelengths of a scene to different angles, and the band-pass filter (9) is used for independently passing the spectral band to be recorded in the +1 level with the highest brightness of the blazed grating (8) and shielding the light of other bands and other grating levels; at the moment, the light dispersed by the blazed grating (8) is converged on the plane where the micro-lens array (10) is located again through the third 4F relay lens (7.3), the light with different wavelengths is focused at the focal length F of the micro-lens behind the micro-lens array (10), the continuous spectrum is spread in a word along the grating dispersion direction, and the spread image is re-imaged on the sensor array of the second gray camera (11.2) through the fourth 4F relay lens (7.4); in the whole light path system, the front light path and the rear light path corresponding to emergent light need to be matched in numerical aperture, namely, the difference between the light projected onto the micro lens array (10) and the numerical aperture of the micro lens array (10) is not more than 5 percent so as to avoid image overlapping and confusion;

by adopting the structure, the reflected light completely records the high-resolution two-dimensional space information of the biological sample under the broad-spectrum white light source (1) in the first gray camera (11.1);

in the emergent light, because the micro-lens array (10) performs sampling segmentation on imaging in a visual field, the imaging of different spectrum channels can be focused in different pixel coordinates, and pixels at corresponding positions in sub-pixels are selected to be recombined to obtain corresponding spectrum information.

2. A rapid spectral microscopy imaging device as defined in claim 1 wherein a is 30%.

3. The method for realizing the rapid spectral microscopic imaging device according to claim 1 or 2, characterized by comprising the following steps:

the method comprises the following steps: an observation object on an objective table (2) is illuminated by a broad-spectrum white light source (1), and a real image of the observation object is divided into two beams by an imaging lens of a microscope objective (3) through a beam splitter prism (4):

the reflected light images the sample on the plane of a first field diaphragm (5.1), and the sample is irradiated on a first gray camera (11.1) through a first 4F relay lens (7.1) and recorded;

emergent light images a sample on a plane where a second field diaphragm (5.2) is located, the sample is emitted to one side of a groove of a blazed grating (8) through a second 4F relay lens (7.2), an aperture diaphragm (6) is used for controlling the numerical aperture of incident light, and at the moment, a real image of an observed object is superposed with the groove surface of the blazed grating (8);

step two: the real image of an observed object mapped on the surface of the blazed grating (8) is subjected to chromatic dispersion, light in a lambda 1-lambda 2 wave band in a grating +1 level passes through the band-pass filter (9) independently, and is converged on the micro-lens array (10) again through the third relay lens (7.3);

step three: because the real image of the observation object mapped on the surface of the blazed grating 8 has a dispersion angle, the real images of different wavelengths re-converged on the micro lens array (10) have different exit angles, and the dispersion occurs along one dimension on the micro lens back focal plane (12), and the real image on the micro lens back focal plane (12) is imaged on the pixel array of the second gray scale camera (11.2) through the fourth 4F relay lens (7.4);

step four: each micro lens in the micro lens array (10) corresponds to a sub-pixel (13) area in the pixel array of the second gray scale camera (11.2), emergent light passing through the micro lens is projected to a middle row of pixels of the sub-pixels (13), at the moment, L pixels at corresponding positions of the middle row in the sub-pixels (13) are recombined, namely, a low-resolution single-wavelength spectrum image corresponding to the wavelength of an object observed on the object stage (2) can be obtained, and the recombined low-resolution single-wavelength spectrum image is obtainedThe spectral images of the single wavelength are respectively P₁、P₂……P_LObtaining L images, wherein the pixel size of the L images is determined by the number of complete micro-lens arrays in the view field of the second gray camera (11.2);

the number of complete micro-lens arrays in the field of view of the second gray scale camera (11.2) is MxN, and the pixel size of the single-wavelength image is MxN;

step five: the spectral response curve of the second gray scale camera (11.2) is divided into L parts so that the L-2 parts of the width of the middle part is the same, and the 1 st and L-th parts of the spectral response curve and the middle part satisfy the following conditions: let the width of the middle part be W_mThe widths of the 1 st and L th parts of the spectral response curve are set as W_e,W_e∈(0.5W_m,W_m]At the moment, R, G, B three groups of spectral response curve graphs corresponding to the second gray camera (11.2) are divided into L parts, and the summation value is multiplied by L to be used as the RGB channel assignment weight; three groups of weighted values are respectively marked as A^R、A^G、A^BWherein the content of the first and second substances,

step six: low resolution single wavelength spectral image P to be recombined_iThe pixel matrix point of (a) is multiplied by the weight value corresponding to the wave band iObtaining the ith waveband image P_iCorresponding RGB three-channel weight matrix

Step seven: the field of view range of the first greyscale camera (11.1) is kept consistent with the full microlens array range that the second greyscale camera (11.2) is able to capture, and then the image Q of the first greyscale camera (11.1) is set to L photographs P of the second greyscale camera (11.2)₁、P₂……P_LThe size of the pixel is L multiplied by L, the number of the sub-pixels which can be captured by the second gray scale camera (11.2) is M multiplied by N, the sub-pixels need M multiplied by N multiplied by L multiplied by the ith wave band image P in the first gray scale camera (11.1)_iCorresponding RGB three-channel weight matrixObtaining three imagesCombining three images as three channels of an RGB image into an RGB image with the pixel size of M multiplied by N multiplied by L, and assigning the same RGB three-channel weight to each sub-pixel as a reference spectral image Q of the ith wave band_i。

4. The method of claim 3, wherein L is an odd integer.

5. The method of claim 4, wherein L-7.

Technical Field

The invention relates to the field of microscopic imaging and spectral imaging, in particular to a rapid spectral microscopic imaging device.

Technical Field

Compared with the traditional imaging technology, the spectrum imaging can record the spectrum information of a scene while shooting a two-dimensional image of the scene, and record the two-dimensional space and the one-dimensional spectrum information. The spectrum imaging technology can increase the richness of recorded information and is beneficial to later analysis and processing. In the initial stage of the spectral imaging technology, the conventional method for acquiring spectral information was used, i.e. two-dimensional spatial information and spectral information at corresponding wavelengths were recorded through a narrow-band filter. The method has the advantages of high precision and easy realization, and has the defects that only a plurality of limited spectral channel information can be obtained, and the spectral information is not coherent. Meanwhile, the method cannot record spectral information on different spectral channels at the same time, so that only spectral imaging of a static scene can be realized.

The rapid spectral microscopic imaging technology can realize the acquisition of a plurality of spectral channels, so that the spectral data is richer. Therefore, the rapid spectral microscopic imaging technology can effectively solve the problems that the spectrum channels in the early spectral imaging technology are few and the dynamic scene image acquisition cannot be processed.

The invention content is as follows:

aiming at the defects in the prior art, the invention provides a rapid spectral microscopic imaging device. The method can simultaneously record high-resolution two-dimensional spatial information and low-resolution one-dimensional spectral information, and can be used for recording spectral microscopic imaging of a dynamic scene.

A rapid spectrum microscopic imaging device comprises a broad spectrum white light source (1), an objective table (2), a microscope objective (3) and a beam splitter prism (4) which are arranged in sequence; a first field diaphragm (5.1), a first 4F relay lens (7.1) and a first gray camera (11.1) are arranged along the direction of the reflected light of the beam splitter prism (4); the device comprises a second field diaphragm (5.2), a second 4F relay lens (7.2), a blazed grating (8), a band-pass filter (9), a third 4F relay lens (7.3), a micro-lens array (10), a fourth 4F relay lens (7.4) and a second gray camera (11.2), wherein the second field diaphragm is arranged along the emergent light direction of a light splitting prism (4).

An aperture diaphragm (6) is arranged between the two groups of lenses of the second 4F relay lens (7.2).

The broad spectrum white light source (1) is used for illuminating an observation object, namely a sample, on the objective table (2), the microscope objective (3) is used for acquiring two-dimensional image information of the sample on the objective table (2) and dividing the two-dimensional image information into two beams of emergent light and reflected light after passing through the beam splitter prism (4):

the reflected light is imaged on a plane where a first field diaphragm (5.1) is located at a brightness of a, and a real image of the first field diaphragm surface is relayed to the plane where a first gray-scale camera (11.1) is located through a first 4F relay lens (7.1);

emergent light is focused on a plane where a second field diaphragm (5.2) is located at the brightness of 1-a, a real image of the second field diaphragm surface is relayed to the surface of a blazed grating (8) through a second 4F relay lens (7.2), and an aperture diaphragm (6) is used for controlling the numerical aperture of incident light. The blazed grating (8) can disperse the transmitted light of different spectral wavelengths of a scene to different angles, and the band-pass filter (9) can independently pass the spectral band to be recorded in the +1 level with the highest brightness of the blazed grating (8) so as to shield the light of other bands and other grating levels. At the moment, the light dispersed by the blazed grating (8) is converged on the plane where the micro-lens array (10) is located again through the third 4F relay lens (7.3), the light with different wavelengths is focused at the focal length F of the micro-lens behind the micro-lens array (10), the continuous spectrum is spread in a word along the grating dispersion direction, and the spread image is re-imaged on the sensor array of the second gray camera (11.2) through the fourth 4F relay lens (7.4). In the whole light path system, the front light path and the rear light path corresponding to emergent light need numerical aperture matching, namely, the difference between the light projected onto the micro lens array (10) and the numerical aperture of the micro lens array (10) is not more than 5 percent, so that image overlapping and confusion are avoided.

By adopting the structure, the reflected light completely records the high-resolution two-dimensional space information of the biological sample under the broad-spectrum white light source (1) in the first gray camera (11.1);

in the emergent light, because the micro-lens array (10) performs sampling segmentation on imaging in a visual field, the imaging of different spectrum channels can be focused in different pixel coordinates, and pixels at corresponding positions in sub-pixels are selected to be recombined to obtain corresponding spectrum information.

Further, a is 30%.

The invention has the following beneficial effects:

the invention realizes the recording of three-dimensional spectral data in a two-dimensional pixel space through pixel partition multiplexing by a unique light path design, namely, the single exposure acquisition of a three-dimensional spectral image can be realized, and the two channels respectively acquire a high-spatial resolution image and a low-resolution spectral image, thereby compensating the spatial resolution loss caused by pixel partition multiplexing to a certain extent;

the device can acquire scene spectrum data in real time and display the scene spectrum data in real time, and the data acquisition and data processing processes have no time delay;

the device can obtain the spectral data of the dynamic scene, and the light source brightness is moderate, so that photobleaching and other damages to biological samples cannot be caused.

Drawings

FIG. 1 is a block diagram of a spectral microimaging apparatus according to the present invention;

FIG. 2 is a graph of the spectral response of a camera according to an embodiment of the present invention;

the system comprises a 1-broad spectrum white light source, a 2-objective table, a 3-microobjective, a 4-beam splitter prism, a 5.1-first field diaphragm, a 5.2-second field diaphragm, a 6-aperture diaphragm, a 7.1-first 4F relay lens, a 7.2-second 4F relay lens, a 7.3-third 4F relay lens, a 7.4-fourth 4F relay lens, an 8-blazed grating, a 9-band-pass filter, a 10-microlens array, an 11.1-first grayscale camera, an 11.2-second grayscale camera, a 12-microlens array focal plane and 13-sub-pixels.

Detailed Description

The invention discloses a spectral microscopic imaging device, which is characterized in that a plurality of continuous spectral information of a biological sample is acquired by single exposure, and the spectral microscopic imaging device comprises the following steps:

referring to fig. 1, the device for spectral microimaging according to the embodiment of the present invention includes a broad spectrum white light source 1, a stage 2, a microscope objective 3, and a beam splitter prism 4, which are arranged in sequence; the first field diaphragm 5.1, the first 4F relay lens 7.1 and the first gray camera 11.1 are arranged along the direction of the light reflected by the beam splitter prism 4; the second field diaphragm 5.2, the second 4F relay lens 7.2, the blazed grating 8, the band-pass filter 9, the third 4F relay lens 7.3, the micro-lens array 10, the fourth 4F relay lens 7.4 and the second gray scale camera 11.2 are arranged along the emergent light direction of the beam splitter prism 4. An aperture stop 6 is arranged between the two sets of lenses of the second 4F relay lens 7.2.

The realization method comprises the following steps:

the method comprises the following steps: the wide spectrum white light source 1 illuminates an observed object on the objective table 2, and the imaging lens of the microscope objective 3 divides the real image of the observed object into two beams through the beam splitter prism 4:

the reflected light images the sample on the plane of the first field diaphragm 5.1, and the sample is incident on the first gray scale camera 11.1 through the first 4F relay lens 7.1 and is recorded;

the emergent light images a sample on a plane where a second field diaphragm 5.2 is located, the sample is emitted to one side of a groove of the blazed grating 8 through a second 4F relay lens 7.2, the aperture diaphragm 6 is used for controlling the numerical aperture of the incident light, and at the moment, the real image of the observed object is superposed with the groove surface of the blazed grating 8.

Step two: the real image of the observation object mapped on the surface of the blazed grating 8 is dispersed, and light of a lambda 1-lambda 2 wave band in the grating +1 level passes through the band-pass filter 9 and is converged on the micro-lens array 10 again through the third relay lens 7.3.

Step three: since the real image of the observation object mapped on the surface of the blazed grating 8 has a dispersion angle, light with different wavelengths is re-converged on the real image of the microlens array 10 to have a different exit angle, and is dispersed along one dimension on the microlens back focal plane 12, and the real image on the microlens back focal plane 12 is imaged on the pixel array of the second gray scale camera 11.2 through the fourth 4F relay lens 7.4.

Step four: each microlens in the microlens array 10 corresponds to a pixel in the pixel array of the second grayscale camera 11.2One sub-pixel 13 region, and the emergent light passing through the micro-lens will be projected onto the middle row of pixels of the sub-pixel 13, at this time, the L pixels at the corresponding position of the middle row in the sub-pixel 13 are recombined, that is, the low resolution single wavelength spectrum image corresponding to the observed object on the object stage 2 under the wavelength can be obtained, and the recombined low resolution single wavelength spectrum images are respectively P₁、P₂……P_LA total of L images are obtained, the pixel size of which is determined by the number of complete microlens arrays in the field of view of the second grayscale camera 11.2.

Further, L is an odd number;

further, L ═ 7.

Further, the number of complete microlens arrays in the field of view of the second grayscale camera 11.2 is M × N, and the single-wavelength image pixel size is M × N.

Step five: the spectral response curve of the second gray scale camera 11.2 is divided into L parts so that the L-2 parts of the width of the middle part is the same, and the 1 st and L-th parts of the spectral response curve and the middle part satisfy the following conditions: let the width of the middle part be W_mThe widths of the 1 st and L th parts of the spectral response curve are set as W_e,W_e∈(0.5W_m,W_m]At this time, the R, G, B three groups of spectral response curves corresponding to the second gray scale camera 11.2 are divided into L parts, and the summation value is multiplied by L to be used as the RGB channel assignment weight. Three groups of weighted values are respectively marked as A^R、A^G、A^BWherein the content of the first and second substances,

step six: low resolution single wavelength spectral image P to be recombined_iThe pixel matrix point of (a) is multiplied by the weight value corresponding to the wave band iObtaining the ith waveband image P_iCorresponding RGB three-channel weight matrix

Step seven: the field of view range of the first greyscale camera 11.1 is kept in line with the full microlens array range that the second greyscale camera 11.2 is able to capture, and then the image Q of the first greyscale camera 11.1 is set to L photographs P of the second greyscale camera 11.2₁、P₂……P_LThe size of the pixel is L × L times, the number of sub-pixels that can be captured by the second grayscale camera 11.2 is M × N, the sub-pixels need M × N × L pixels to record, and the corresponding M × N sub-pixels with the size of L × L in the first grayscale camera 11.1 are multiplied by the ith band image P_iCorresponding RGB three-channel weight matrixObtaining three imagesCombining three images as three channels of an RGB image into an RGB image with the pixel size of M multiplied by N multiplied by L, and assigning the same RGB three-channel weight to each sub-pixel as a reference spectral image Q of the ith wave band_i。