CN115937080A - Hexagonal lattice illumination super-resolution microscopic system and image reconstruction method - Google Patents
Hexagonal lattice illumination super-resolution microscopic system and image reconstruction method Download PDFInfo
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Abstract
The invention belongs to the technical field of microscopes, and provides a hexagonal lattice illuminated super-resolution microscope system and an image reconstruction method, wherein the hexagonal lattice illuminated super-resolution microscope system comprises: a digital micromirror device comprising a plurality of micromirrors configured in a hexagonal array for reflecting illumination light to modulate said illumination light into hexagonal structured light; and the central control unit is configured to control the digital micromirror device to adjust the micromirror reflection angle according to a specific time sequence so as to generate a plurality of phase-shifted images, and reconstruct the image stack after the image acquisition of the camera detection module is completed so as to output a super-resolution image. The invention has the advantages that the hexagonal structured light is adopted, shooting in three directions is not needed like the traditional super-resolution structured light illumination, the number of single-phase images required by one super-resolution image is reduced in principle, and parallel operation is carried out under a GPU, so that the time required by super-resolution image reconstruction is prolonged, and the reconstruction frame rate reaches the standard required by real-time reconstruction.
Description
Technical Field
The invention relates to the technical field of microscopes, in particular to a hexagonal lattice illumination super-resolution microscope system and an image reconstruction method.
Background
In a conventional wide field fluorescence microscope, unless the sample is very flat, all other images are affected by fluorescence emission within a certain error from the longitudinal depth of the microscope objective focal plane. Structured-illumination microscopes provide a better way to eliminate this unwanted image effect by projecting a linear grid onto the sample. The method needs to shoot images of different phases at different positions of an online grid, and can obtain super-resolution images from a certain number of single-phase images by adopting a matched reconstruction algorithm.
In recent years, most conventional approaches have taken a modulation pattern that employs a grid of lines in a single direction for structured lighting. In the two-dimensional SIM super-resolution reconstruction algorithm provided by Gustafsson, at least 9 original images are required to be input into one super-resolution image, the 9 images are divided into 3 groups, each group is provided with three corresponding phase shift images in three illumination directions, the super-resolution reconstruction method adopts 3 linear equation sets to solve and shift frequency, and finally resolution improvement in each direction is obtained. The traditional reconstruction method is limited by the influence of the response speed of a system or the processing speed of a reconstruction algorithm, the frame rate of super-resolution image reconstruction is low, the reconstruction speed of a video frame is low, and the requirement of real-time reconstruction is difficult to achieve. In order to increase the speed of the super-resolution reconstruction algorithm, some new methods can adopt original single-phase images with 4 images as low as input for reconstruction, but when structural light is constructed, the robustness of an initial phase is low, and a phase value needs to be set with higher precision when a system is constructed.
Disclosure of Invention
The invention aims to provide a microscope technology for performing structured light modulation and image stack super-resolution reconstruction based on a DMD (digital micromirror device), which can reconstruct a super-resolution image by only 7 single-phase images in a hexagonal structured light mode so as to solve the problems.
In order to achieve the purpose, the invention adopts the technical scheme that:
a hexagonal lattice illuminated super-resolution microscope system at least comprises an illumination light path module and a camera detection module, wherein illumination light emitted by a multi-color laser arranged in an illumination light path generates fluorescence on a sample to be detected and is collected by the camera detection module, and the system comprises:
a digital micromirror device comprising a plurality of micromirrors configured in a hexagonal array to reflect illumination light to modulate said illumination light into hexagonal structured light; and the central control unit is configured to control the digital micromirror device to adjust the micromirror reflection angle according to a specific time sequence so as to generate a plurality of phase-shifted images, and reconstruct the image stack after the image acquisition of the camera detection module is completed so as to output a super-resolution image.
Further, a dichroic mirror is disposed between the camera detection module and the digital micromirror device, and configured to reflect the emitted light emitted from the digital micromirror device to a sample to be detected to excite fluorescence, and the excited fluorescence is transmitted to the camera detection module by the dichroic mirror to perform image acquisition.
Furthermore, a tube lens and a first optical filter are sequentially arranged between the digital micromirror device and the dichroic mirror along a light transmission path, the tube lens enters the microscope frame, stray light is filtered by the first optical filter, and then the structured light generated by the digital micromirror device is transmitted to the dichroic mirror.
Furthermore, a second optical filter and a converging lens are sequentially arranged between the dichroic mirror and the camera detection module along a light transmission path, and the fluorescent light transmitted by the dichroic mirror passes through the second optical filter to filter light rays with wavelengths outside the exciting light, and is focused on the camera detection module by the converging lens for collection.
Furthermore, the illumination light path module comprises an objective lens, a pentagonal prism, a double-cemented lens and a reflector which are sequentially arranged in the front and back order of a light source transmission path, besides the multicolor laser; the objective lens expands the beam of the illumination light excited by the multicolor laser, the illumination light passes through the size of the folding space of the pentagonal prism, the parallel light beams emitted after passing through the objective lens are converged at the double-cemented lens, and the reflector adjusts the angle of the light rays incident on the digital micromirror device so that the excited illumination light is incident on the hexagonal array in the digital micromirror device.
Further, the wavelength of the illumination light emitted by the multi-color laser is adjusted by the central control unit.
The invention also provides a reconstruction method of the output image of the hexagonal lattice illumination super-resolution microscope system, which comprises the following steps:
s1, emitting illumination light with preset wavelength by a multi-color laser controlled by a central control unit to be incident on a reflection surface of a digital micro-mirror device along a light transmission path, and projecting the illumination light on a sample to be measured to form hexagonal lattice illumination light after the illumination light is reflected and modulated by a hexagonal array of the digital micro-mirror device;
s2, the central control unit controls the inclination direction of the hexagonal array on the digital micro-mirror device on the CPU platform, so that at least 7 original image stacks which are collected by the camera detection module and perform hexagonal lattice projection modulation monophasic images according to a specific time sequence are obtained, and the original image stacks are transmitted into a GPU from the CPU platform;
s3, the central control unit resamples the obtained original image stack in the GPU, and the original image stack is processed through a pre-filter generated by combining an optical transfer function and an attenuation coefficient for inhibiting an out-of-focus signal;
s4, estimating initial parameters of the lattice structure light modulation image from the original image information, and calculating and determining a spatial frequency vector, a spatial phase and a modulation amplitude by adopting a cross-correlation algorithm;
s5, calculating a reconstruction factor in a GPU (graphics processing Unit) by using a parallel operation mode, and enabling the reconstruction factor and a pre-filter to jointly act on an original image stack processed by the pre-filter to perform frequency shift and reconstruction processing on the image;
s6, substituting the reconstruction factor into a wiener filter constructed by an optical transfer function, an attenuation coefficient and a pre-filter together, and performing post-filtering on the reconstructed image;
and S7, transmitting the data information in the GPU back to the CPU platform, and sorting the data information of the super-resolution image so as to output the reconstructed super-resolution image.
Compared with the prior art, the invention at least comprises the following beneficial effects:
(1) Through adopting hexagonal structure light, need not shoot on three direction like traditional super-resolution structured light illumination, the modulation of hexagonal structure light itself can carry out even modulation to the sample in 3 directions on the spatial plane, reduces the quantity of the required single-phase picture of a super-resolution image in principle to promote super-resolution image reconstruction algorithm's speed, greatly reduced super-resolution image required original image's the number of times of shooting. Meanwhile, three-direction lighting modes are not needed, and the complexity of the system is further reduced;
(2) The 7 single-phase images in the hex-SIM are equivalently and linearly converted into 9 images of a conventional demodulation algorithm, and the hexagonal lattice algorithm is processed in parallel in a GPU frame, so that the single-phase images are transferred to a new position in a Fourier space, the resolution of the system is increased, the time required by super-resolution image reconstruction is greatly reduced from the aspects of programs and algorithms, and the reconstruction frame rate reaches the standard required by real-time reconstruction;
(3) The DMD is used as a spatial modulator to modulate light incident on the surface of the DMD to generate structured light, high-speed capture of images is realized according to the high frame rate of the structured light, a super-resolution 3D data set is recorded, the switching time between two adjacent single-phase images is greatly reduced, and the artifact effect of a sample to be detected in the shooting process is reduced;
(4) The opening number of DMD micromirror pixel units with reflection modulation function can be set on micrometer level as required while independently performing binary control on millions of micromirror arrays on the DMD, so that the accuracy of structured light can be enhanced, the period size of the structured light can be freely adjusted, the flexibility of sample object modulation is enhanced, and the contrast of sample modulation can be adjusted;
(5) Required light rays in incident light rays are reflected through the opening and closing states of pixels in a specific period of the DMD, other light rays are efficiently filtered, mechanical disturbance caused by the fact that a mechanical motor is used for adjusting a mask in the prior art is avoided, and imaging precision is improved;
drawings
FIG. 1 is a schematic diagram of a hexagonal lattice illuminated super-resolution microscope system according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a hexagonal structured light configuration in an embodiment of the present invention;
FIG. 3 is a schematic diagram of DMD modulation to form structured light illumination in an embodiment of the present invention;
FIG. 4 is a flow chart of the hexagonal lattice illumination reconstruction to generate a super-resolved image in an embodiment of the present invention;
in the figure, 1, a multicolor laser, 2, an optical fiber, 3, an objective lens, 4, a pentagonal prism, 5, a double-cemented lens, 6, a reflector, 7, a digital micromirror device, 8, a tube mirror, 9, a first optical filter, 10, a dichroic mirror, 11, a high-power objective lens, 12, a sample, 13, a second optical filter, 14 and a converging lens; 15. camera detection module, 16, central control unit.
Detailed Description
It should be noted that all the directional indicators (such as upper, lower, left, right, front, and rear … …) in the embodiment of the present invention are only used to explain the relative position relationship between the components, the motion situation, and the like in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indicator is changed accordingly.
Moreover, descriptions of the present invention as relating to "first," "second," "a," etc. are for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicit ly indicating a number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless explicitly specified otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "connected," "secured," and the like are to be construed broadly, and for example, "secured" may be a fixed connection, a removable connection, or an integral part; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In addition, the technical solutions in the embodiments of the present invention may be combined with each other, but it must be based on the realization of those skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination of technical solutions should not be considered to exist, and is not within the protection scope of the present invention.
The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described with reference to the drawings, but the present invention is not limited to these embodiments.
As shown in fig. 1, the hexagonal lattice illuminated super-resolution microscope system provided by the present invention includes an illumination light path module composed of a multi-color laser 1, an optical fiber 2, an objective lens 3, a pentagonal prism 4, a double cemented lens 5 and a reflector 6.
An optical fiber 2 is connected at the exit of the polychromatic laser 1, an objective lens 3 is located behind the optical fiber 2 and the end face of the optical fiber 2 is located at the front focal plane of said objective lens 3. Wherein, the illuminating light emitted by the multicolor laser 1 is expanded by the objective lens 3 in turn, the expanded light path is folded by the pentagonal prism 4, the folded light path is focused by a double-cemented lens 5, and then is focused on a receiving surface of a Digital Micromirror Device (DMD) 7 after the deflection angle is adjusted by a reflecting mirror 6.
The illumination light is modulated by the digital micro-mirror device 7 to generate structured light, enters the microscope frame through the tube mirror 8, is reflected by the dichroic mirror 10 after the stray light is filtered by the first optical filter 9, and is projected to a sample 12 to be measured through the high power objective lens 11. The structured light and the sample to be detected coated with the fluorescent dye are excited to generate exciting light, the exciting light returns along the sample to be detected 12 and the high power objective lens 11, the light with the wavelength outside the exciting light is filtered by the second optical filter 13 after being transmitted by the dichroic mirror 10, and finally, the light is focused on a sensing receiving part of the camera detection module 15 by the converging lens 14.
The central control unit 16 can control the multicolor laser 1 to emit illumination light with different wavelengths, and the digital micro-mirror device 7 is loaded by the central control unit 16 according to specific time sequence to generate the required modulation image of the structured light with different phase shifts before being modulated by the digital micro-mirror device 7.
The timing of the exposure of the CCD camera in the camera detection module to the images produced by the excitation light is also controlled by the central control unit 16 before recording by the camera detection module 15.
The digital micromirror device 7 has millions of micromirror units, each of which can be controlled by a central control unit independently for binarization, and the size of the micromirror unit is in the order of micrometers, and the high-precision micromirror unit can construct a hexagonal lattice pattern with higher resolution.
As shown in fig. 2, P is the period length of the hexagonal cell in the structured light, and is related to the periodic rule converted to the frequency spectrum. The higher the precision of the digital micromirror device 7, the larger the space in which the hexagonal pitch P can be adjusted in the figure, and the easier the construction of the hexagonal lattice.
As shown in fig. 3, the micromirror element in the digital micromirror device 7 can be adjusted to two states of "0" and "1", the "0" state representing the "off" state and the "1" state representing the "on" state. The binary input of the central control unit 16 can determine the state of the reflective surface of each micromirror in the digital micromirror device 7.
When the square micro-mirror unit is in an 'on' state, the square micro-mirror unit deflects by +12 degrees around a symmetry axis inclined by 45 degrees, and reflected light is received by a subsequent system; in the "off" state, the square micromirror cell is deflected by-12 degrees about a 45-degree oblique axis of symmetry, and the reflected light escapes out of the system as stray light.
In a specific timing sequence, different modulation images loaded on the digital micromirror device 7 are as shown in the bottom of fig. 4, each time the image moves in the same direction and size, and after 7 times of movement, the 8 th sub-modulation image is the same as the 1 st sub-modulation image. At the time of the different T values, the central control unit 16 binarizes the array from the input image so that the reflected light is subsequently received as a different phase shift modulation effect, thereby generating the specific structured light. The central control unit controls the modulation image time sequence of the digital micro-mirror device 7 to be matched with the time sequence of a monophase picture shot by a CCD camera to generate 7 pairs of continuous phase-shifted images, and the 7 pairs of images are one period, so that super-resolution images are output through a reconstruction algorithm in the central control unit subsequently.
According to the above embodiment of the present invention, the present invention further provides a reconstruction method of an output image of a hexagonal lattice illumination super-resolution microscope system, as shown in fig. 3, which includes the steps of:
s1, emitting illumination light with preset wavelength by a multi-color laser controlled by a central control unit to be incident on a reflection surface of a digital micro-mirror device along a light transmission path, and projecting the illumination light on a sample to be measured to form hexagonal lattice illumination light after the illumination light is reflected and modulated by a hexagonal array of the digital micro-mirror device;
s2, the central control unit controls the inclination direction of the hexagonal array on the digital micromirror device on the CPU platform, so that at least 7 original image stacks which are collected by the camera detection module and used for carrying out hexagonal lattice projection modulation single-phase diagrams according to a specific time sequence are obtained, and the original image stacks are transmitted into a GPU from the CPU platform;
the size of the periodic structure of the hexagonal lattice image is determined by the parameter of the light modulation depth of the structure finally projected on the sample surface. The periodic structure of the hexagonal lattice illumination light field mathematically satisfies the following equation:
where x and y represent coordinate positions of a two-dimensional spatial plane, and p is a periodic feature of a hexagonal periodic structure (a periodic pitch of hexagonal cells).
After simplification, the product can be sorted into a form proportional to the index:
the hexagonal structured light is projected onto the image and the first phase shifted image can be determined by the following expression:
there are seven unknowns in the above formula, each being I 0 、I s1 、I s2 、I s3 、
And &>
It can be seen that at least 7 monophasic images are required to resolve all the corresponding unknown parameters to obtain a super-resolution image.
The central control unit controls the multicolor laser to be switched on and switched off according to a specific time sequence on the CPU platform, controls the digital micro-mirror device to load images required by hexagonal lattice illumination modulation according to the specific time sequence, controls the digital micro-mirror device to output signals to the CCD camera according to the specific time sequence, and controls the CCD camera to carry out image acquisition operation under the time sequence output by the digital micro-mirror device.
In order to ensure that the system has relatively long image exposure time when in continuous imaging, the central control unit adopts a trigger mode of synchronous reading, and the calculation formula of the frame rate is as follows:
wherein M is a frame rate, the unit is fps, M is a camera set starting line number, and H is the length of the error time delay of a camera system.
And then recording at least 7 original image stacks of hexagonal lattice projection modulation single-phase diagrams in sequence, and transferring the image stacks into a GPU from a CPU platform to prepare for subsequent parallel operation.
S3, the central control unit resamples the obtained original image stack in the GPU, and the original image stack is processed through a pre-filter generated by combining an optical transfer function and an attenuation coefficient for inhibiting an out-of-focus signal;
in a CUDA (computer Unified Device Architecture) library in a GPU framework, resampling an input original image stack, storing image data units and GPU storage units in a one-to-one correspondence mode, completing a control trigger part of hardware in a CPU platform, completing a pure operation part required in image reconstruction in a GPU, and generating a prefilter to act on the original image stack by combining a system optical transfer function constructed in the GPU and an attenuation coefficient for inhibiting an out-of-focus signal.
S4, estimating initial parameters of the lattice structure light modulation image from the original image information, and calculating and determining a spatial frequency vector, a spatial phase and a modulation amplitude by adopting a cross-correlation algorithm;
the initial intensity information for the image stack is:
estimating system initial parameters of lattice structure light modulation image from initial intensity information of image stack to obtain spatial frequency vector
Spatial phase size->
And modulation amplitude size phi m . Space(s)The phase is greater or less>
In the estimation of (2), an autocorrelation reconstruction method is adopted for facilitating parallel operation, and the relationship is as follows:
where a is the intensity coefficient of its component.
S5, using a parallel operation mode in the GPU and obtaining a space frequency vector
Spatial phase size->
And modulation amplitude size phi m Substituting the parameters into the equation for 4 frequency bands:
and (3) obtaining a reconstruction factor of the system:
the reconstruction factor and the pre-filter jointly act on the original image stack processed by the pre-filter to perform frequency shift and reconstruction processing on the image.
S6, substituting the reconstruction factor into a wiener filter constructed by an optical transfer function, an attenuation coefficient and a pre-filter, and performing post-filtering on the reconstructed image to eliminate artifacts caused by sample movement;
and S7, transmitting the data information in the GPU back to the CPU platform, and sorting the data information of the super-resolution image so as to output the reconstructed super-resolution image.
The invention linearly converts 7 images into 9 images of a conventional demodulation algorithm equivalently, processes the hexagonal lattice demodulation reconstruction algorithm in the GPU in parallel, thereby transferring the images to a new position in a Fourier space and increasing the resolution of the system.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments, or alternatives may be employed, by those skilled in the art, without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (7)
1. A hexagonal lattice illumination super-resolution microscope system at least comprises an illumination light path module and a camera detection module, wherein illumination light emitted by a multi-color laser arranged in an illumination light path generates fluorescence on a sample to be detected and is collected by the camera detection module, and the hexagonal lattice illumination super-resolution microscope system is characterized by comprising:
a digital micromirror device comprising a plurality of micromirrors configured in a hexagonal array for reflecting illumination light to modulate said illumination light into hexagonal structured light; and the central control unit is configured to control the digital micromirror device to adjust the micromirror reflection angle according to a specific time sequence so as to generate a plurality of phase-shifted images, and reconstruct the image stack after the image acquisition of the camera detection module is completed so as to output a super-resolution image.
2. The hexagonal-lattice-illuminated super-resolution microscope system according to claim 1, wherein a dichroic mirror is disposed between the camera detection module and the digital micro-mirror device, and configured to reflect the emitted light from the digital micro-mirror device to a sample to be measured to excite fluorescence, and the excited fluorescence is transmitted from the dichroic mirror to the camera detection module for image acquisition.
3. The hexagonal-lattice-illuminated super-resolution microscope system according to claim 2, wherein a tube lens and a first optical filter are sequentially disposed between the digital micromirror device and the dichroic mirror along a light transmission path, the tube lens enters a microscope frame, and the first optical filter filters stray light and then transmits structured light generated by the digital micromirror device to the dichroic mirror.
4. The hexagonal lattice illuminated super-resolution microscope system according to claim 2, wherein a second optical filter and a converging lens are sequentially disposed between the dichroic mirror and the camera detection module along a light transmission path, and the fluorescent light transmitted by the dichroic mirror passes through the second optical filter to filter out light with a wavelength outside the excitation light, and is focused by the converging lens on the camera detection module for collection.
5. The hexagonal lattice illuminated super-resolution microscope system according to claim 1, wherein the illumination light path module comprises an objective lens, a pentagonal prism, a double cemented lens and a reflector, which are sequentially arranged in the front-rear order of a light source transmission path, in addition to the multicolor laser; the objective lens expands the beam of the illumination light excited by the multicolor laser, the illumination light passes through the size of the folding space of the pentagonal prism, the parallel light beams emitted after passing through the objective lens are converged at the double-cemented lens, and the reflector adjusts the angle of the light rays incident on the digital micromirror device so that the excited illumination light is incident on the hexagonal array in the digital micromirror device.
6. A hexagonal-lattice illuminated super-resolution microscope system according to claim 1 wherein the wavelength of the illumination light emitted by the multi-color laser is adjusted by the central control unit.
7. A reconstruction method of hexagonal lattice illuminated super-resolution microscope system output images is characterized by comprising the following steps:
s1, emitting illumination light with preset wavelength by a multi-color laser controlled by a central control unit to be incident on a reflection surface of a digital micro-mirror device along a light transmission path, and projecting the illumination light on a sample to be measured to form hexagonal lattice illumination light after the illumination light is reflected and modulated by a hexagonal array of the digital micro-mirror device;
s2, the central control unit controls the inclination direction of the hexagonal array on the digital micromirror device on the CPU platform, so that at least 7 original image stacks which are collected by the camera detection module and used for carrying out hexagonal lattice projection modulation single-phase diagrams according to a specific time sequence are obtained, and the original image stacks are transmitted into a GPU from the CPU platform;
s3, the central control unit resamples the obtained original image stack in the GPU, and the original image stack is processed through a pre-filter generated by combining an optical transfer function and an attenuation coefficient for inhibiting an out-of-focus signal;
s4, estimating initial parameters of the lattice structure light modulation image from the original image information, and calculating and determining a spatial frequency vector, a spatial phase and a modulation amplitude by adopting a cross-correlation algorithm;
s5, calculating a reconstruction factor in a GPU (graphics processing Unit) by using a parallel operation mode, acting the reconstruction factor and a pre-filter on an original image stack processed by the pre-filter together, and performing frequency shift and reconstruction processing on the image;
s6, substituting the reconstruction factor into a wiener filter constructed by an optical transfer function, an attenuation coefficient and a pre-filter together, and performing post-filtering on the reconstructed image;
and S7, transmitting the data information in the GPU back to the CPU platform, and sorting the super-resolution image data information so as to output a reconstructed super-resolution image.
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