Structured light high space-time resolution off-axis digital holographic three-dimensional chromatography systemTechnical Field
The invention belongs to the technical field of optical microscopic measurement, and particularly relates to a structured light high-space-time resolution off-axis digital holographic three-dimensional chromatography system which is used for carrying out non-contact and high-precision three-dimensional chromatography on a transmission sample.
Background
Digital holography (Digital Holography Microscopy, DHM) uses the principles of interference and diffraction to record the amplitude and phase information of the light waves and reconstruct the three-dimensional morphology of the sample by phase demodulation. Off-axis digital holographic microscopy combines the advantages of optical interference and microscopic imaging, and records a single optical hologram in discrete digitized form on an optoelectronic recording device such as a CCD or CMOS by one exposure. And then, carrying out numerical simulation and digital reproduction by a computer to obtain the three-dimensional appearance of the measured object. The technology has the remarkable advantages of high imaging speed, longitudinal nano-scale resolution, high full-field quantification, no need of marking and the like.
However, the lateral resolution of off-axis digital holographic microscopy is limited by the diffraction limit of wide-field imaging, and it is difficult to fully resolve the fine features and sharp contours of sub-wavelength microstructures. In addition, although DHM can realize quantitative phase imaging, unlike the three-dimensional morphology phase measurement of a reflective micro-nano structure, the phase information obtained by measuring a biological sample once is only accumulation of refractive index of light on a propagation path, and the internal three-dimensional structure of a transparent sample cannot be resolved.
With the continuous and deep research of biomedicine, the spatial resolution and the time resolution of an imaging system are urgently required to be improved so as to observe finer subcellular structures and dynamic changes thereof, thereby providing key technical support for revealing the motion mechanism of the subcellular structures. Similarly, the sub-wavelength industrial device generally has a micro-nano characteristic structure far smaller than the wavelength of light, which puts higher requirements on the high space-time resolution microscopy technology to acquire the micro-structure characteristic information such as the critical dimension and the like, and provides technical guarantee for the optimal design and performance control of the device.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a structured light high space-time resolution off-axis digital holographic three-dimensional chromatography system. The method comprises the steps of encoding a high-frequency signal to a low-frequency area, improving the stability of matching of a DMD (Digital Micromirror Device ) and a system, improving the transverse resolution to two times, generating new structural light annular multi-angle illumination, realizing three-dimensional diffraction chromatography of transparent samples such as biological cells, and visualizing the three-dimensional structures inside the samples, and improving the spatial resolution and the time resolution of imaging the biological cells and sub-wavelength devices by digital holographic microscopy in a multi-layer slice diagram, an envelope diagram, a continuous chromatographic diagram and other display modes.
The invention is realized by the following technical scheme:
the invention provides a structured light high space-time resolution off-axis digital holographic three-dimensional chromatography system, which comprises a light source generation and collimation module, a polarization beam splitting module, a spatial light modulation module, a spatial filtering module, a sample illumination microscopy module, an image acquisition module and a data analysis and display module, wherein:
the light source generating and collimating module is used for emitting laser and filtering and collimating;
the polarization light splitting module is used for converting the light after filtering and collimation into object light and reference light in two mutually perpendicular different polarization directions;
the spatial light modulation module is used for performing wavefront modulation on object light by adopting a DMD device to generate structural light for illuminating a sample so as to realize annular multi-angle illumination on the sample, wherein the rotation angle of the DMD device along a z-axis and the inclination angle of an xy-axis are adjustable;
The spatial filtering module is used for filtering the structure light after wave front modulation;
The sample illumination microscopic module is used for illuminating the sample by adopting modulated and filtered structured light, and the light illuminating the sample is modulated by the sample into object light carrying sample phase information;
the image acquisition module is used for acquiring interference fringes of reference light and object light carrying sample phase information to obtain a hologram;
The data analysis and display module is used for processing the hologram by adopting a three-dimensional chromatography and display algorithm and generating a continuous chromatography, an envelope and a multi-layer slice of the sample.
Preferably, the light source generating and collimating module comprises a laser diode, a first micro objective lens, a first diaphragm and a first double-cemented lens, wherein the first micro objective lens, the first diaphragm and the first double-cemented lens are sequentially arranged along a laser light path.
The invention is characterized in that the polarization beam splitting module comprises a first polarization beam splitting prism, a first polarizing plate, a first reflecting mirror and a second diaphragm, wherein a transmission light path of the first polarization beam splitting prism is an object light path, a reflection light path of the first polarization beam splitting prism is a reference light path, light of the object light path and light of the reference light path are perpendicular to each other and have different polarization directions, the first polarizing plate and the first reflecting mirror are sequentially positioned on the reference light path, the polarizing angle of the first polarizing plate is the same as the vibration angle of light reflected by the first polarization beam splitting prism, and the second diaphragm is positioned on the object light path.
The invention is characterized in that the spatial light modulation module comprises a second reflector, a DMD device and a DMD connection adjusting piece, wherein light emitted from a light path of the polarized light splitting module enters the DMD device through the second reflector, and the DMD device is arranged on the DMD connection adjusting piece.
The DMD connecting and adjusting piece comprises an adjusting rear plate, adjusting bolts, adjusting springs and a DMD connecting plate, wherein the DMD connecting plate is provided with a pair of arc-shaped track clamping groove structures, each arc-shaped track clamping groove structure comprises an arc-shaped track and a plurality of bolt clamping grooves communicated with the arc-shaped track, the bolt clamping grooves corresponding to the arc-shaped tracks form different connecting angles, the DMD device is connected with the arc-shaped track clamping groove structure on the DMD connecting plate through bolts and is adjustable in connecting angle, the connecting angle corresponds to the rotating angle of the DMD along the z axis, the adjusting rear plate is connected with the DMD connecting plate through three adjusting springs, the two adjusting bolts are arranged along the diagonal of the adjusting rear plate, one adjusting spring is arranged near each adjusting bolt, and one adjusting spring is further arranged between the two adjusting bolts, and the distance between the adjusting rear plate and the DMD connecting plate can be changed by loosening or screwing the two adjusting bolts, and the inclination angle of the DMD device along the xy axis can be adjusted.
Preferably, the spatial filtering module comprises a second double-cemented lens, a third diaphragm, a third double-cemented lens, a third reflector and a second polarizer, and forms a 4f filtering system, wherein the second double-cemented lens and the third double-cemented lens have the same focal length, the center of the DMD device in the object light path is positioned on the front focal point of the second double-cemented lens, and the center of the third diaphragm is positioned on the rear focal point of the second double-cemented lens and simultaneously positioned on the front focal point of the third double-cemented lens.
As the preferable mode of the invention, the third diaphragm is an iris diaphragm, and three orders in the light wave spectrum modulated by the DMD device can completely pass through the third diaphragm by adjusting the third diaphragm, wherein the three orders are respectively 0 order, +1 order and-1 order.
The sample illumination micro-module comprises a fourth double-cemented lens, a second micro-objective lens, a sample stage, a third micro-objective lens and a fifth double-cemented lens, wherein the front focal point of the fourth double-cemented lens is overlapped with the rear focal point of the third double-cemented lens in the space filtering module, the focal length of the third double-cemented lens is equal to that of the fourth double-cemented lens, the front focal point of the third micro-objective lens is overlapped with the rear focal point of the second micro-objective lens, the sample stage is placed at the position where the focal points of the second micro-objective lens and the third micro-objective lens are overlapped, and the front focal point of the fifth double-cemented lens is overlapped with the rear focal point of the third micro-objective lens.
The image acquisition module comprises a first unpolarized beam-splitting prism and a CCD camera, wherein the CCD camera is placed on the back focal plane of a fifth double-cemented lens in the sample illumination microscopic module and is conjugate with the position of a sample to be detected, object light is transmitted through the first unpolarized beam-splitting prism, reference light is reflected through the first unpolarized beam-splitting prism, the object light and the reference light meet to generate interference to obtain a hologram, and the off-axis angle between the object light and the reference light can separate each level in the spectrum of the hologram.
As the preferable mode of the invention, the data analysis and display module comprises a host and a display, wherein a three-dimensional chromatography and display algorithm is arranged in the host, a CCD camera transmits holograms to the host, and the holograms are calculated by the three-dimensional chromatography and display algorithm to obtain continuous chromatography, envelope and multi-layer slice.
Compared with the prior art, the invention has the advantages that:
The invention aims to provide a structured light high space-time resolution off-axis digital holographic three-dimensional chromatography system, which constructs a high resolution digital holographic microscopic system by generating structured light annular multi-angle illumination by utilizing a Digital Micromirror Device (DMD) and realizes three-dimensional diffraction chromatography of a sample to be detected. The system fully plays the characteristics of non-contact, rapidness and no need of dyeing of the digital holographic technology, and improves the transverse resolution to twice that of the prior art. In addition, the spatial filtering technology based on the 4f system is adopted, so that the contrast of the finally generated hologram is obviously improved, and the accuracy of sample phase reconstruction is ensured. The design of the DMD connection adjusting piece further enhances the matching stability of the DMD and the system, and can accurately adjust the rotation angle of the DMD on the z axis and the inclination angle of the xy axis, so that the requirements of different light paths are met, and structural light is ensured to be emitted into a subsequent system module in multiple angles.
The system is particularly applied to biological cell imaging. The three-dimensional diffraction tomography device can reproduce the three-dimensional structure and refractive index distribution inside cells without longitudinal scanning, thereby realizing high-resolution imaging and multi-angle illumination. The technology remarkably improves the resolving power of biological samples, and enables observation of finer cell structures and dynamic changes thereof to be possible, thereby providing key technical support for revealing the motion mechanism of subcellular structures.
In the imaging aspect of sub-wavelength industrial devices, the system can break through the diffraction limit of optical microscopy, and accurately represent micro-nano characteristics and clear contours of the surfaces of the devices. The method provides necessary technical foundation for optimizing design and performance control of the sub-wavelength device, and has wide application potential in the micro-nano precise structure measurement field.
Drawings
FIG. 1 is a schematic diagram of a structured light high spatial-temporal resolution off-axis digital holographic three-dimensional chromatography system according to an embodiment of the present invention;
FIG. 2 is a structured light illumination pattern;
FIG. 3 is a schematic diagram of a three-dimensional model and parts of a DMD connection adjuster, wherein (a) the DMD connection plate, (b) the adjustment back plate, and (c) the combination of the connection adjuster and the DMD;
FIG. 4 is an illustration of a digital holographic resolution limit calibration, wherein (a) a conventional digital holographic resolution limit calibration, and (b) a structured light digital holographic resolution limit calibration;
FIG. 5 is a diagram of a multi-slice and an envelope, wherein (a) the multi-slice and (b) the envelope;
FIG. 6 is a continuous chromatogram showing (a) layer 1, (b) layer 24, and (c) layer 48;
FIG. 7 is a flow chart illustrating the operation of the system between the modules shown in the present embodiment;
In FIG. 1, 1-laser source, 2-first microscope objective, 3-first diaphragm, 4-first doublet, 5-first polarization beam splitter, 6-first polarizer, 7-first mirror, 8-second diaphragm, 9-second mirror, 10-DMD device, 11-second doublet, 12-third diaphragm, 13-third doublet, 14-third mirror, 15-second polarizer, 16-fourth doublet, 17-second microscope objective, 18-sample stage, 19-third microscope objective, 20-fifth doublet, 21-first non-polarization beam splitter, 22-CCD camera, 23-host and display, 24-adjustment back plate, 25-adjustment bolt, 26-adjustment spring, 27-DMD connection plate.
Detailed Description
Further details are provided below with reference to the specific embodiments.
The foregoing and other features, aspects and principles of the present invention are apparent from the following detailed description of the invention when considered in connection with the accompanying drawings. The technical means adopted by the invention for achieving the aim can be better understood through the specific description of the embodiment.
The embodiment of the invention provides a structured light high space-time resolution off-axis digital holographic three-dimensional chromatography system, wherein the working flow of the mutual coordination among all modules in the system is shown as a figure 7, the system comprises a light source generation and collimation module, a polarization beam splitting module, a spatial light modulation module, a spatial filtering module, a sample illumination microscopy module, an image acquisition module and a data analysis and display module, wherein a sample to be detected is placed in the sample illumination microscopy module, and the sample meets the light-penetrability condition, such as biological cells, sub-wavelength scale micro-nano chips and the like.
The device comprises a light source generating and collimating module, a polarization light splitting module, a sample illumination module and an image acquisition module, wherein the light source generating and collimating module is used for emitting laser with the wavelength of 632.8nm and filtering and collimating, the polarization light splitting module is used for converting the filtered and collimated light into two mutually perpendicular object light and reference light with different polarization directions, the spatial light modulation module is used for performing wave front modulation on the incident light by adopting a DMD to generate structural light for illuminating a sample, annular multi-angle illumination is realized so as to perform three-dimensional diffraction chromatography on the sample, the DMD connection adjusting piece is used for changing the rotation angle of the DMD along a z-axis (optical axis) and the inclination angle of an xy-axis, the requirements of different optical paths can be met, the different angles of the structural light can be injected into a subsequent system module, the spatial light filtering module is used for filtering the structural light generated after wave front modulation, the sample illumination module is used for irradiating the modulated and filtered incident light to the sample, the incident light is modulated into the object light carrying sample phase information again by the transparent sample, and the image acquisition module is used for acquiring interference fringes to obtain a hologram. The data analysis and display module comprises a host and a display, wherein a three-dimensional chromatography and display algorithm is arranged in the host, and a multi-layer slice diagram, an envelope diagram and a continuous chromatography diagram can be obtained after calculation through the algorithm.
As shown in fig. 1, an embodiment of the present invention provides an optical path diagram of a structured light high spatial-temporal resolution off-axis digital holographic three-dimensional chromatography system, which includes a laser diode 1, microscope objectives 2, 17, 19, diaphragms 3, 8, 12, two cemented lenses 4, 11, 13, 16, 20, polarizers 6, 15, a polarizing beam splitter prism 5, a non-polarizing beam splitter prism 21, mirrors 7, 9, 14, a sample stage 18 to be tested, a dmd device 10, a ccd camera 22, a host and a display 23.
The light source generating and collimating module comprises a laser light source 1, a first micro objective lens 2, a first diaphragm 3 and a first double-cemented lens 4, wherein the first micro objective lens 2, the first diaphragm 3 and the first double-cemented lens 4 are arranged along a light path.
In this embodiment, the wavelength of the laser light source 1 is 632.8nm, the first micro objective lens 2 is used for limiting and converging the visible light bandwidth generated by the laser light source 1, the diaphragm 3 is used for spatially filtering the light focused by the micro objective lens 2 to improve the time coherence of the light source and filtering part of stray light to improve the imaging quality of the final hologram, and the first double-cemented lens 4 is used for collimating the light beam filtered by the diaphragm to make the light beam entering the subsequent module be parallel light.
The polarization beam splitting module comprises a first polarization beam splitting prism 5, a first polaroid 6, a first reflecting mirror 7 and a second diaphragm 8.
In this embodiment, the first polarization splitting prism 5 converts the incident light into two light beams with different polarization directions perpendicular to each other, wherein the transmission light path is an object light path, the reflection light path perpendicular to the transmission light path is a reference light path, the first polarizer 6 and the first reflecting mirror 7 are located on the reference light path, the polarization angle of the first polarizer 6 is the same as the vibration angle of the light beam reflected by the first polarization splitting prism 5, so as to obtain polarized reference light with stricter polarization, the first reflecting mirror 7 reflects the light transmitted through the first polarizer 6, so that the light energy of the reference light path and the light energy of the object light path meet in the image acquisition module to generate interference, and the interference light is guaranteed to have a certain off-axis angle, and the second diaphragm 8 is located on the object light path, so as to spatially filter the light of the object light path, and prepare for the subsequent incident light adjustment to the DMD.
The spatial light modulation module comprises a second mirror 9, a DMD device 10 and a DMD connection adjustment.
In this embodiment, the light emitted from the second diaphragm 8 of the polarization beam splitter module is reflected to the DMD device 10 by the second mirror 9, and the installation angle of the second mirror 9 needs to ensure that the light emitted from the DMD device 10 can enter the center of the subsequent optical path, and since the deflection angle of the DMD device 10 is 17.5 ° when in operation, the included angle between the mirror surface of the second mirror 9 and the plane of the DMD device 10 should be 27.5 °. The DMD device is based on semiconductor manufacturing technology and is composed of a high-speed digital light reflective switch array, for example, the array pattern loaded by the DMD device 10 is a checkerboard pattern as shown in fig. 2, rotation and translation are performed on the basis of the checkerboard pattern, and the total pixels of the checkerboard pattern are required to be 1000×1000, wherein the pixels of each checkerboard are 20×20. The model diagram and the part diagram of the DMD connection adjusting member are shown in fig. 3, and the DMD connection adjusting member comprises an adjusting rear plate 24, an adjusting bolt 25, an adjusting spring 26 and a DMD connecting plate 27, wherein fig. 3 (a) is a front view of the DMD connecting plate, fig. 3 (b) is each view of the adjusting rear plate, and fig. 3 (c) is a three-dimensional model diagram of the connection adjusting member and the DMD combination. The DMD connection adjusting piece is used for adjusting the rotation angle of the DMD along the z axis (optical axis) and the inclination angle of the xy axis. The DMD device 10 can be connected with a circular arc track slot structure designed on the DMD connecting plate 27 through bolts, when the rotation angle of the DMD device along the z axis (optical axis) needs to be adjusted, the bolts need to be unscrewed, the bolts enter the circular arc track along the bolt slots, the DMD device is rotated, the bolts enter the bolt slots again by selecting a proper angle, the DMD is fixed on the DMD connection adjusting piece by fastening the bolts, and in the embodiment, the rotation angle of the DMD along the z axis (optical axis) can be adjusted to 30 °, 60 ° and 90 °. The adjusting rear plate 24 and the DMD connecting plate 27 are connected with two adjusting bolts through three adjusting springs, wherein the two adjusting bolts are arranged along the diagonal line of the adjusting rear plate 24, one adjusting spring is arranged near each adjusting bolt, and one adjusting spring is arranged between the two adjusting bolts. By loosening/tightening the two adjusting bolts, the distance between the adjusting rear plate 24 and the DMD connecting plate 27 can be changed, and the inclination angle of the DMD device 10 along the xy axis can be adjusted, and in this embodiment, the inclination angle adjustment range along the xy axis is 0 to 3.5 °. The adjusting rear plate 24 can be firmly connected with the optical platform, so that the optical platform can be matched with an optical path.
The spatial filtering module comprises a second double-cemented lens 11, a third diaphragm 12, a third double-cemented lens 13, a third reflector 14 and a second polarizer 15, and forms a 4f filtering system.
In this embodiment, the center of the DMD device 10 in the object light path is located at the front focal point of the second double-cemented lens 11, the center of the third diaphragm 12 is located at the rear focal point of the second double-cemented lens 11, and at the same time, is located at the front focal point of the third double-cemented lens 13, the second double-cemented lens 11 is used for converging light beams, so that the light beams can filter out unwanted light beam portions through the third diaphragm 12, and the second double-cemented lens 13 can convert the converged light beams into parallel light beams to enter the subsequent light path. The third diaphragm 12 is an iris diaphragm, and by adjusting the diaphragm, three orders of the light wave modulated by the DMD device 10 can completely pass through the diaphragm, namely, 0 order, +1 order and-1 order, so as to prepare for annular light illumination required by subsequent three-dimensional chromatography, and preferably, the focal lengths of the two double cemented lenses are equal. The third reflecting mirror 14 reflects the light transmitted through the third double-cemented lens 13 to the second polarizer, and the polarization direction of the second polarizer 15 is identical to the transmission polarization direction of the first polarization splitting prism 5 in the polarization splitting module, so as to obtain polarized object light with stricter polarization state.
The sample illumination microscopy module comprises a fourth doublet 16, a second microscope objective 17, a sample stage 18, a third microscope objective 19 and a fifth doublet 20.
In this embodiment, the pattern modulated by the DMD device is conjugated to the surface of the sample on the sample stage 18 through the fourth double-cemented lens 16 and the second double-cemented lens 17 by using the confocal principle of the 4f filtering system of the spatial filtering module, the beam modulated by the sample is injected into the subsequent optical path in the form of parallel beam through the third double-cemented lens 19 and the fifth double-cemented lens 20, the beam at this time already carries the phase information of the sample, the front focal point of the fourth double-cemented lens 16 coincides with the back focal point of the third double-cemented lens 13 in the spatial filtering module, the front focal point of the third double-cemented lens 19 coincides with the back focal point of the second double-cemented lens 17, the sample stage 18 is placed at the position where the focal points of the second double-cemented lens 17 and the third double-cemented lens 19 coincide, and the front focal point of the fifth double-cemented lens 20 coincides with the back focal point of the third double-cemented lens 19.
The fourth 16 and fifth 20 cemented doublets preferably have equal focal lengths, the second microscope objective 17 is used to focus and illuminate the sample, the third microscope objective 19 is used to magnify the image of the sample, and the third microscope objective 19 preferably has a higher numerical aperture.
The image acquisition module comprises a first unpolarized beam-splitting prism 21 and a CCD camera 22, wherein the CCD camera 22 is used for acquiring interference fringes generated by the first unpolarized beam-splitting prism 21 to obtain a hologram.
It should be noted that the CCD camera 22 needs to be placed on the back focal plane of the fifth double-cemented lens 20 in the sample illumination micro-module and conjugated with the sample to be measured, the first unpolarized beam splitter prism 21 is located between the fifth double-cemented lens 20 and the CCD camera 22, the light on the object light path is transmitted through the first unpolarized beam splitter prism 21, the light on the reference light path is reflected through the first unpolarized beam splitter prism 21, the two light beams meet at a certain off-axis angle to generate interference, the interference fringes are recorded by the CCD camera 22, and the off-axis angle can separate the levels (level 0, +1 and level-1) in the obtained interference pattern spectrum.
The data analysis and display module comprises a host and a display 23, wherein a three-dimensional chromatography and display algorithm is built in the host, the CCD camera 22 transmits holograms to the host, and continuous chromatography, envelope and multi-layer slice can be obtained and displayed through algorithm calculation.
As shown in fig. 4, which is a resolution limit calibration diagram of a digital holographic system, in an optical microscope, the diffraction limit is a physical limit that assumes that the aberration-free optical system can reach the maximum imaging resolution, and for a noncoherent imaging system, the diffraction limit is expressed as a rayleigh Li Panju:
Where λ is the illumination light source wavelength, and na=nsinθ represents the numerical aperture of the optical system. However, the above formula is only applicable to incoherent imaging systems, and for digital holography and other coherent imaging systems, the above formula should be rewritten as:
The system uses a light source with the wavelength of 632.8nm, the numerical aperture NA of an optical system is 0.6, the resolution limit is about 864.8nm obtained by substituting the numerical aperture NA into the formula (2), the traditional digital hologram can only be resolved to the 9 th group 2 element line pair of the high-resolution standard plate after experiments, the corresponding line width is 870nm, as shown in the figure 4 (a), the maximum resolution of the 10 th group 2 element line pair can be found after structural light illumination and algorithm demodulation reconstruction, as shown in the figure 4 (b), the corresponding line width is 435nm, and the resolution of the verification system is improved to be twice.
Fig. 5 (a) shows a diagram of the obtained multi-layer slice, the x and y axes are pixel coordinates of the obtained image, the z axis is the number of slice layers, which is 16-34 layers, the color bands on the right side are relative refractive indexes, which indicate that the internal structure of the cell shows different relative refractive indexes in different slice planes, fig. 5 (b) shows a diagram of the obtained envelope, which is a three-dimensional combination of the same data, the x and y axes are pixel coordinates of the obtained image, the z axis also represents the number of slice layers, and the three-dimensional morphology and internal structure of the cell can be intuitively seen.
As shown in fig. 6, which is a graph of the obtained continuous chromatogram, fig. 6 (a), fig. (b) and fig. 6 (c) show the relative refractive index graphs when the number of obtained slice layers is 1 layer, 24 layers and 48 layers, respectively, wherein the x and y axes are the pixel coordinates of the obtained image, z is the number of slice layers, and different colors show different relative refractive indexes.
The foregoing is merely an example of the present application and common knowledge of the characteristics and the like of a scheme is not described in detail herein. It should be noted that the above-mentioned embodiments are merely illustrative, and not restrictive, and that it will be apparent to those skilled in the art that various changes and modifications can be made herein without departing from the application as defined by the appended claims. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.