CN115755364A - Multi-mode synchronous imaging microscope and method based on illumination wavelength and direction coding - Google Patents
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Abstract
The invention discloses a multimode synchronous imaging microscope and a method based on illumination wavelength and direction coding. The device comprises a programmable full-color LED dot matrix lighting control system, an XYZ-axis motion objective table, a lens module and an imaging observation module. The LED array is placed below the sample stage as the light source for the microscope imaging system, centered on the optical axis of the system. By changing the illumination mode of the LED array, bright field imaging, dark field imaging, phase contrast imaging and differential phase contrast imaging can be realized, wherein the color coding illumination mode can realize multi-mode real-time synchronous imaging without mode switching. The Fourier spectrum of the rear focal plane of the lens module is regulated and controlled, and color and direction coding and decoding can be carried out on the image. The imaging optical path has a simple structure, can flexibly realize optical microscopic imaging in various modes, allows the free regulation and control of the dimensions such as space, color and the like of a focal plane behind a light source and an objective lens, and can realize multi-channel real-time information acquisition by a color camera.
Description
Technical Field
The invention relates to the technical field of optical microscopic imaging, in particular to a multimode synchronous imaging microscope device based on illumination wavelength and direction coding and a method thereof.
Background
With the continuous development of science and technology, the human beings continuously and deeply explore the micro world, and the requirements on microscope imaging are continuously improved. The optical microscopic imaging technology has the characteristics of real time, high resolution, non-invasiveness and the like, and the imaging scale of the optical microscopic imaging technology can span cells, tissues and even life bodies, thereby greatly expanding the understanding boundary of people on the essence of life. Particularly in the aspect of cell biology, how to clearly and accurately observe organelles is always an important requirement in life science and biomedical research. Therefore, microscopic imaging techniques have been the focus of research in the optical field.
Currently, optical microscopes have been provided with multiple imaging modalities for a variety of different types of research. The existing imaging comprises bright field, dark field, phase contrast, polarization, fluorescence, laser scanning confocal and the like, wherein bright field, dark field and phase contrast microscopic imaging are still the three most widely used microscopic imaging modes at present. By combining three microscopic imaging modes of bright field, dark field and phase contrast, various spectrum information of the unmarked biological sample can be presented qualitatively and the imaging contrast is improved. However, the current commercial microscope cannot realize the three imaging modes at the same time, and an additional imaging element is required.
The programmable LED array is a low-cost and simple spatial light modulation device, and is widely applied to the field of microscopes to realize illumination modulation. At present, a plurality of researches adopt an LED array to simultaneously realize bright field, dark field, differential phase contrast and other imaging functions. The mercury lamp for illumination in the existing commercial microscope is replaced by a programmable LED array which can be controlled by a computer, the wavelength of incident light is regulated and controlled by regulating and controlling illumination color, different patterns are designed and displayed to control the illumination direction, multiple illumination modes can be easily realized, the light path does not need to be regulated, the light path structure is greatly simplified, and the cost is reduced. At least three images are needed for realizing bright field imaging, dark field imaging and differential phase contrast imaging, in order to achieve the effect of real-time imaging, a time division multiplexing method is generally adopted to control the synchronization of LED array illumination pattern switching and camera image acquisition, but the method can influence the acquisition rate of a sample for the sample with a high motion speed. The color coding LED array is combined with a color camera, so that the problem can be well solved, mode switching is not needed, the color camera only needs to shoot a color image and extract three channels of RGB (red, green and blue) of the image, and then bright field, dark field and differential phase contrast imaging information can be synchronously acquired.
In addition, because the structure of the current commercial microscope is closed and free operation can not be carried out on the rear focal plane of the objective lens, the traditional commercial microscope does not have the functions of simultaneously allowing the illumination wavelength and the illumination mode of the light source to be freely regulated and controlled, freely regulating and controlling the space spectrum of the rear focal plane of the objective lens and freely regulating and controlling the channel of the color camera into a whole.
Disclosure of Invention
In view of the above-mentioned circumstances of the prior art, it is an object of the present invention to provide a multimode synchronous imaging microscope apparatus based on illumination wavelength and direction encoding and an operating method thereof, which can simultaneously realize multimode imaging such as bright field, dark field, differential phase contrast, etc. without switching an objective lens. Because the back focal plane of the objective lens is exposed in the air, and different filters are arranged on the back focal plane, the invention can realize the functions of the Zernike phase contrast microscope and various spatial filtering experiments, change the imaging spatial frequency spectrum of a sample and extract different information. Meanwhile, the invention can simultaneously extract the information of the red, green and blue three-band of the color image for analysis. The microscope with the simple structure and the simple operation and the multi-dimensional free regulation and control of the illumination wavelength and the illumination direction provides detailed visual data for biomedical research, and plays an extremely important role in promoting the development of cell biology.
In order to achieve the purpose, the invention adopts the technical scheme that:
the multi-mode synchronous imaging microscope based on the illumination wavelength and the direction code comprises an illumination control system, a moving object carrying system, a lens module and an imaging observation module, wherein the illumination control system comprises a programmable full-color LED dot matrix light source which is used for controlling the illumination direction and the illumination color; the moving object carrying system comprises a displacement table which can freely move in a three-dimensional space, and a microscope slide holder is arranged on the displacement table and used for fixing and regulating the imaging position of a sample; the lens module captures light rays from the sample surface, and a Fourier spectrum surface is arranged on the back focal plane of the lens module for optical filtering; the imaging observation module shoots a color image, RGB three channels of the color image are separated, signals of different color channels are extracted for processing, and three images containing different information are obtained.
Furthermore, the LED lattice light source is an LED flexible screen which allows a program to control the illumination pattern, the illumination wavelength and the brightness of pixel points, so that the switching of different illumination modes and the illumination color coding are realized.
Furthermore, a transmission type lighting mode is adopted, the light source generates light and then irradiates the sample to be measured, and the light source is uniformly distributed around the axis of the lens.
Furthermore, the Fourier frequency spectrum surface of the lens module is positioned in the external space of the lens module to allow space frequency spectrum control, a space filter is arranged on the Fourier frequency spectrum surface to change the space frequency spectrum structure of the object image and screen frequency spectrum information in different directions, the lens captures light from the sample surface, and the distribution of plane waves carrying the sample information on the rear focal plane of the lens group is in direct proportion to the Fourier transform of the distribution of the sample.
Further, the original image shot by the color camera can be subjected to RGB three-channel separation.
Further, the lens module comprises an objective lens, and the Fourier spectrum plane is located on the back focal plane of the objective lens.
Furthermore, a mask pattern is placed on the back focal plane, and image filtering is performed.
The method for controlling by using the device comprises the following specific steps:
(1) And controlling the LED pixel dot matrix flexible screen of the WS2812B series by a program, and controlling the light emitting position, the light emitting quantity, the light emitting color and the light emitting brightness of the LED particles according to the requirement of an illumination pattern so as to control the illumination wavelength, the illumination angle and direction and the illumination intensity.
(2) Under the condition of not changing an objective lens, obtaining corresponding bright field optical, dark field optical and differential phase contrast images by changing the illumination mode of the LED array; the phase contrast microscope objective is characterized in that an annular phase plate is added on the back focal plane of the objective on the basis of a dark field microscope objective mode, and the diameter of a light source incidence annular region is matched with that of the back focal plane annular phase plate region of the microscope objective. A high-pass/low-pass/band-pass/directional filtering spatial filter and the like are arranged on a rear focal plane of the objective lens and a Fourier spectrum plane of the system, so that optical information processing is realized. The illumination mode is color coded, so that bright field optical, dark field optical and differential phase contrast images can be synchronously obtained.
(3) Putting a sample to be observed on a sample objective table, fixing a sample glass slide by using a microscope glass slide clamp, adjusting the sample coordinates in the XY direction to enable the sample to be positioned in the center of a visual field, and adjusting the sample position in the Z direction to obtain a clear image.
(4) An RGB camera of a microscope imaging system is adopted to shoot images and display the images on a display, and information of three channels of a color image is extracted.
The invention uses the LED array as a light source and the large-format lens as an objective lens, thereby realizing multi-mode synchronous microscopic imaging. The invention has the following beneficial effects:
(1) The invention realizes multi-mode microscopic imaging by flexibly controlling the incident illumination of the light source, changes the illumination wavelength and direction under the condition of not changing the objective lens, and synchronously obtains the multi-mode microscopic image, thereby realizing the microscopic function which can not be realized or is difficult to realize by adopting the traditional microscope.
(2) The invention does not need the sample to be dyed, and can observe various dyed and undyed samples.
(3) In the invention, the Fourier imaging surface is just at the back focal surface of the objective lens, so that some mask patterns can be placed on the back focal surface for image filtering.
(4) The LED array illumination pattern switching and the color camera multi-channel image acquisition are synchronous, and real-time imaging can be realized.
Drawings
FIG. 1 is a schematic structural diagram of a multimode synchronous imaging microscope device based on illumination wavelength and direction encoding in an embodiment of the present invention. The system comprises an LED pixel dot matrix flexible screen 1-WS2812B, a sample stage 2, an objective lens group 3, a Fourier back focal plane 4 and a color camera 5.
Fig. 2 is a light path diagram in the embodiment of the present invention.
FIG. 3 is a schematic diagram of a multi-mode imaging implementation in an embodiment of the invention.
Fig. 4 is a schematic diagram of the optical path of bright field mode illumination in an embodiment of the present invention.
FIG. 5 is a schematic optical path diagram of dark field mode illumination in an embodiment of the present invention.
FIG. 6 is a schematic diagram of the optical path of the differential phase contrast microscopy imaging mode in an embodiment of the invention.
FIG. 7 is a schematic diagram of the optical path of color coded illumination in an embodiment of the present invention.
Fig. 8 is a schematic optical path diagram of spatial filtering regulation in the embodiment of the present invention.
FIG. 9 is a schematic diagram of the optical path of the phase-contrast microscopy imaging mode in an embodiment of the present invention.
Detailed Description
The following detailed description is to be read in connection with the accompanying drawings and the detailed description.
Referring to fig. 1, the multimode synchronous imaging microscope based on illumination wavelength and direction encoding of the present embodiment includes an illumination control system, a moving object system, a lens module, and an imaging observation system. The lighting control system comprises a program-controlled WS2812BLED pixel lattice flexible screen 1. The moving object carrying system comprises a manual triaxial flexible displacement platform and a microscope fixing clamp which can freely move in an XYZ-axis three-dimensional space, the manual triaxial flexible displacement platform and the microscope fixing clamp are fixedly connected together through screws, and a biological sample glass slide is fixed under a clamp spring clamp to form a sample object carrying platform 2. The centers of the sample stage 2, the objective lens group 3, and the color camera 5 are maintained in a straight line, wherein light transmitted through the sample stage 2 is collected by the objective lens group 3 and is magnified to be imaged at the focal plane of the color camera 5. The imaging observation system comprises a color camera 5 with a high-resolution image sensor, which is connected with a computer host and a display for displaying the imaging result.
The WS2812B LED pixel dot matrix flexible screen 1 is used as an illumination light source of the multi-mode microscope, and is an intelligent external control LED light source integrating a control circuit and a light-emitting circuit. It is placed directly under the sample stage 2 with a loading distance between 10-25cm and the WS2812BLED pixel lattice flexible screen l centered on the optical axis of the objective lens group 3 and the color camera 5. The individual LED elements in the LED array are three-color red, green and blue LEDs, with typical wavelengths of red 625nm, green 525nm and blue 465nm. A plurality of LED lamp beads can be connected in series through a signal line, and the control of at most 256 LEDs can be realized at the refresh frequency of 30 Hz. Each element is a pixel. The pixel point internally comprises an intelligent digital interface data latch signal shaping amplification driving circuit, a high-precision internal oscillator and a programmable constant current control part, and the color height consistency of pixel point light is effectively ensured. LED specific characteristic parameters are given in table 1. In the LED array, 16 rows and 16 columns of LED bulbs are shared, and 256-level brightness display can be realized by three primary colors of each pixel point.
TABLE 1WS2812BLED array of characteristic parameters
A control method of a multimode synchronous imaging microscope based on illumination wavelength and direction encoding comprises bright field imaging, dark field imaging, differential phase contrast imaging and color encoding illumination imaging capable of synchronously realizing the above microscopic modes. Different optical elements are placed on the Fourier spectrum surface 4 of the device, so that a spatial filtering experiment can be carried out, the device is applied to optical information processing, and a phase plate required by phase contrast imaging is a special spatial filter, so that the device can also realize Zernike phase contrast imaging. Bright field imaging, dark field imaging and differential phase contrast imaging are realized in similar steps, the difference is only that WS2812B LED pixel lattice flexible screen l displays different patterns, and the bright field imaging, the dark field imaging and the differential phase contrast imaging can be simultaneously obtained through color coding illumination by specific pattern design and color control. Based on the Zernike phase contrast imaging, a phase retardation plate is added on the back focal plane of the objective lens group 3. A spatial filter is arranged on a Fourier spectrum surface 4 behind the objective lens group 3, so that the image quality is improved, and the optical information processing is realized.
The invention relates to an operating method of a multimode synchronous imaging microscope based on illumination wavelength and direction coding, which comprises the following steps:
the method comprises the following steps: the patterns required for bright field, dark field, differential phase contrast microscopy, phase contrast microscopy and color coded illumination are displayed in the LED array 1.
Step two: under the configuration, a sample to be observed is placed on the sample object stage 2, the position of the sample is adjusted to enable the image to be clear, and a microscopic imaging result is shot in real time by the color camera 5.
Step three: under the configuration, a spatial filter can be placed on the back focal plane of the objective lens, the position of the sample is adjusted to enable the image to be clear, and the image is shot by the color camera 5 to obtain an imaging result after optical filtering.
Referring to fig. 2, illumination light is projected on a sample stage 2, transmitted light and scattered light enter an objective lens group 3, and the objective lens group 3 magnifies an image and is finally collected by a color camera 5. By utilizing the Fourier transform relationship of the light field distributions on the front focal plane and the rear focal plane of the lens, the spatial frequency spectrums of various images can be analyzed, and the images can be identified and classified.
Referring to fig. 3, the illumination mode of the LED array is changed by program control without changing the objective lens to obtain bright field, dark field, phase contrast, differential phase contrast image, color-coded illumination raw image and image of the sample after filter action. For an LED illumination source fixed in the imaging system, the distance D between the object focal plane and the LED is known, and the numerical aperture NA of the objective lens 0 And = nsin θ, where n is a medium through which the light passes and θ represents a maximum incident angle at which the light can enter the objective lens. The combination relation of the optical axis and the illumination angle
It can be seen that the maximum radius of light that the LED array can enter the objective lens
For the known imaging system, the objective NA 0 Namely, n =1 in the air medium is determined, so that the radius R of the bright field illumination range of the LED array is determined.
Referring to fig. 4, the designated pattern for bright field microscopy contains a colored central circle with no light emission in the rest of the pattern. The illumination wavelength can be selected from any one of red, green and blue lights or a mixed color of three primary colors. The radius of the pattern is slightly smaller than the numerical aperture of the objective lens group 3, and bright field microscopic imaging can be realized.
Referring to fig. 5, the designated pattern corresponding to the dark field microscopic imaging is a hollow colored ring, and the rest of the pattern does not emit light. The circular ring is tangent to the numerical aperture of the objective lens group 3, the illumination wavelength can be any one of red, green and blue lights or the mixed color of three primary colors, and dark field microscopic imaging can be realized.
Referring to fig. 6, a designated pattern corresponding to differential phase contrast microscopy needs to divide a color ring/circle into a plurality of sub-regions with equal areas, the adjacent colors of the sub-regions can be different colors, and the illumination wavelength can be selected from any one of red, green and blue lights or a mixed color of three primary colors. Generally, the LED array is divided into two parts which are mutually symmetrical and complementary left and right or up and down to realize respective illumination, and simultaneously, the camera is controlled to successively acquire two frames of oblique illumination images I 1 And I 2 Then, calculation processing is performed:
wherein, I DPC For differential phase contrast imaging, I 1 +I 2 For bright field imaging. See (a) and (b) in fig. 6 in detail.
Referring to fig. 7, color coded illumination can achieve the above bright field, dark field, differential phase contrast imaging effects simultaneously without illumination pattern mode switching. The sample is illuminated by an illumination model mixed with three wavelengths of red, green and blue, amplified by an objective lens group 3 after passing through the sample, and finally a color image is obtained by a color camera 5. The illumination is complementary to red and blue light within the illumination numerical aperture, and the exterior is illuminated with green light. The red and blue illuminating lights at the center are mixed to form a purple sample background, the detail high-frequency information such as the outline of the sample is marked by green, and the obtained color pattern is similar to the reineger berg imaging effect. The color coding illumination imaging mode can analyze bright field, dark field and differential phase contrast microscopic effects simultaneously by only taking one picture. Wherein bright field imaging is determined by two semi-circle radii of red (R) and blue (B) in the LED screen, and dark field illumination is determined by the outer circle of green (G)And (4) determining the area. The color image shot by the color camera 5 is subjected to RGB three-channel separation to obtain three images, namely a red channel I R Blue channel I B Green channel I G . Wherein I R +I B For bright field imaging, I G Dark field imaging is performed. By the formula
Calculating a differential phase contrast image, I DPC As a result of the differential imaging.
Referring to fig. 8, a spatial filtering experiment was performed using the present apparatus. The designated pattern contains a colored central circle with the remainder non-emitting. The illumination wavelength can be any one of red, green and blue lights or a mixed color of three primary colors. Generally, the pattern radius is slightly smaller than the numerical aperture of the objective lens assembly 3. A filter is placed on the back focal plane of the objective lens group, so that areas in different directions pass through different colors, and then a color image is obtained on an image plane. A-e in fig. 8 are typical filters, and a in fig. 8 is a high-pass filter, which is a light screen opaque in the central part, and filters out low-frequency components in the diffraction spectrum after passing through the sample and allows high-frequency components to pass through, and can be used for highlighting the edge part of the image or realizing contrast inversion of the sample image; b in fig. 8 is a low-pass filter, which is used to filter out high-frequency noise, such as net-like structures in some special samples, by filtering out high-frequency components and passing only low-frequency components near zero frequency; FIG. 8, c, is a band pass filter that passes some of the desired spectral components, the remainder being filtered out and used to remove noise; d-e in fig. 8 is a directional filter that can be used to remove or only pass certain directions of the spectrum, for highlighting certain features of the sample image. In addition, some special filters can be designed, such as gratings in different directions to modulate the image, also called as a theta modulation method, so that different parts in the image are coded with different colors, and thus, optical/image color coding and decoding can be performed.
Referring to fig. 9, in particular, the phase plate required for phase contrast microscopy is a special spatial filter. The designated pattern corresponding to the phase contrast microscopic imaging is a hollow colored ring, and the rest part does not emit light. A phase space filter is placed at the back focal plane, and the phase plate region is matched with the annular region which does not emit light. The illumination wavelength can be selected from any one of red, green and blue lights or the mixed color of three primary colors, and the Zernike phase contrast microscopic effect can be realized.
The system can synchronously acquire the microscopic images in various modes in real time, regulate and control the focal plane behind the objective lens to carry out spatial filtering, and realize optical information processing. The invention regulates and controls the illumination wavelength and the illumination direction of the light source through a program, carries out direction and color coding on the rear focal plane of the objective lens, and simultaneously carries out information extraction on three channels of the color picture, thereby providing a new method for the research of the microscopic image. The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention.
Claims (8)
1. The multi-mode synchronous imaging microscope based on the illumination wavelength and direction coding is characterized by comprising an illumination control system, a moving object carrying system, a lens module and an imaging observation module, wherein the illumination control system comprises a programmable full-color LED dot matrix light source which is used for controlling the illumination direction and the illumination color; the moving object carrying system comprises a displacement table which can freely move in a three-dimensional space, and a microscope slide holder is arranged on the displacement table and used for fixing and regulating the imaging position of a sample; the lens module captures light rays from a sample surface, and a Fourier spectrum surface is arranged on a back focal plane of the lens module for optical filtering; the imaging observation module shoots a color image, RGB three channels of the color image are separated, signals of different color channels are extracted for processing, and three images containing different information are obtained.
2. The illumination wavelength and direction coding based multimode synchronous imaging microscope of claim 1, wherein the LED lattice light source is an LED flexible screen that allows program control of its pixel point illumination pattern, illumination wavelength and brightness to effect switching of different illumination modes and illumination color coding.
3. The multi-mode synchronous imaging microscope based on illumination wavelength and direction coding as claimed in claim 2, characterized in that, by adopting a transmission illumination mode, the light source generates light and then irradiates on the sample to be measured, and the light source is uniformly distributed around the axis of the lens.
4. The microscope of claim 1, wherein the fourier spectrum plane is located outside the lens module to allow spatial spectrum manipulation, a spatial filter is disposed on the fourier spectrum plane to change the spatial spectrum structure of the object image and screen the spectrum information in different directions, the lens captures the light from the sample plane, and the distribution of the plane wave carrying the sample information on the focal plane behind the lens set is proportional to the fourier transform of the sample distribution.
5. The illumination wavelength and direction coding based multimode synchronous imaging microscope of claim 1, wherein the original image taken by the color camera can be RGB three channel separated.
6. The illumination wavelength and direction encoding based multimode synchronous imaging microscope of claim 1, wherein the lens module comprises an objective lens, and the fourier spectrum plane is located at a back focal plane of the objective lens.
7. The illumination wavelength and direction encoding based multimode synchronous imaging microscope of claim 6, wherein a mask pattern is placed on the back focal plane for image filtering.
8. The method of operating a multimode, synchronized imaging microscope based on illumination wavelength and direction encoding as recited in any of claims 1-7, comprising:
step 1: the programmable full-color LED dot matrix light source is controlled by a program, and the light-emitting position, the light-emitting quantity, the light-emitting color and the light-emitting brightness of the LED particles are controlled according to the requirement of an illumination pattern so as to control the illumination wavelength, the illumination angle and direction and the illumination intensity;
and 2, step: under the condition of not changing the lens module, obtaining corresponding bright field optics, dark field optics and differential phase contrast images by changing the illumination mode of the LED array; the phase contrast microobjective is characterized in that an annular phase plate is added at the back focal plane of the objective on the basis of a dark field microobjective mode, and the diameter of a light source incident annular region is matched with that of the back focal plane annular phase plate of the microobjective; a high-pass/low-pass/band-pass/direction filtering spatial filter and the like are arranged on a Fourier frequency spectrum surface of a rear focal plane of the objective lens to realize optical information processing; a color coding illumination mode, wherein bright field optics, dark field optics and differential phase contrast images are synchronously obtained;
and step 3: putting a sample to be observed on a sample stage, fixing a sample glass slide by using a microscope glass slide clamp, adjusting the sample coordinates in the XY direction to enable the sample to be positioned in the center of a visual field, and adjusting the sample position in the Z direction to obtain a clear image;
and 4, step 4: an RGB camera of a microscope imaging system is adopted to shoot images and display the images on a display, and information of three channels of a color image is extracted.
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