Compact dual-wavelength common-path holographic microscope

Abstract

The invention discloses a compact dual-wavelength common-path holographic microscope. Laser beams emitted by two laser light sources are modulated into a vertical polarization laser beam and a horizontal polarization laser beam through a collimation and beam expanding system and a polaroid respectively; beam combination is carried out through a first non-polarization beam splitting prism and a second non-polarization beam splitting prism, and object light and reference light are split through a plate-type beam splitter; the object light irradiates the sample through the microscope objective; the reference light is reflected light passing through the plate-type beam splitter; the laser beams under the two wavelengths are separated after passing through the polarization splitting prism, the reference light and the object light under each wavelength interfere with each other, and a holographic image is recorded by the image sensor. The device has the advantages of being real-time, rapid, compact in arrangement and high in vibration resistance and time stability, separation and angle adjustment are carried out on light beams of reference light, object light and two wavelengths through a simple optical element and an adjustment mode, and the device has obvious advantages on morphology characterization of a precise structure and high-precision dynamic imaging.

Description

Technical Field

[0001] The invention relates to the technical field of digital holographic imaging, in particular to a compact dual-wavelength common-path holographic microscope. Background Art

[0002] Digital holography is the development of common holography, and the technology includes two steps: recording and reproducing. Different from common holography, digital holography uses detectors such as CCD and CMOS instead of holographic plates to record and store holograms. In addition, the reproduction method of digital holography is to obtain the amplitude and phase distribution of the object light field through numerical reconstruction by numerical calculation.

[0003] Digital holographic microscopy is a combination of digital holographic technology and microscopic imaging technology. It is a non-contact, high-quality, high-resolution phase imaging technology that is widely used to measure parameters such as cell thickness, refractive index, and the surface morphology of opaque structures. Digital holographic microscopy has the following advantages:

[0004] (1) Only one picture needs to be taken within the exposure time of the recording camera to obtain the amplitude distribution and phase distribution of the object to be measured;

[0005] (2) It has digital autofocus function and does not require manual mechanical focusing.

[0006] In the application of digital holographic microscopy, when the optical length of the object to be measured is greater than the wavelength of the light source, the phase distribution of the object to be measured will be wrapped between [-π,π], which is called the phase wrapping phenomenon. In order to obtain a continuous and accurate phase distribution, the truncated phase needs to be connected through a phase unwrapping algorithm. However, the maximum measurable optical length between adjacent pixels of the object to be measured is limited to one wavelength. When the object to be measured does not meet this condition, such as for a measurement object with a mutation structure, the unwrapping algorithm will fail and cause phase recovery errors. Therefore, single-wavelength holographic microscopy technology has limitations for the measurement of high-depth mutation structures. Dual-wavelength holographic microscopy is an extension of single-wavelength holographic microscopy. It obtains accurate phase distribution by acquiring phase maps of two wavelengths separately and subtracting them, thereby expanding the longitudinal measurement range of DHM.

[0007] When dual-wavelength holographic microscopy is applied to the morphology measurement of precision structures, the dual-wavelength phase recovery process will lead to the amplification of system noise. As an optical path arrangement with higher stability and stronger vibration resistance than the split optical path system, the combination of the common path system and digital holographic technology is an inevitable trend. However, in the existing dual-wavelength common-path holographic microscopy optical path arrangement, the separation method of reference light and object light and the optical elements used are relatively complicated. Pinhole elements are usually used for spatial filtering (KUMAR M, QUAN X, AWATSUJI Y, et al. Single-shot common-path off-axis dual-wavelength digital holographic microscopy [J / OL]. Applied Optics, 2020, 59 (24): 7144-7152. DOI: 10.1364 / AO.395001.), or multiple reflective elements are used for beam separation (GUO R, WANG F. Compact and stable real-time dual-wavelength digital holographic microscopy with a long-working distance objective [J / OL]. Optics Express, 2017, 25 (20): 24512-24520. DOI: 10.1364 / OE.25.024512; SHAN M, LIU L, ZHONG Z, et al. Single-shot dual-wavelength off-axis quasi-common-path digital holography using polarization-multiplexing [J / OL]. Optics Express, 2017, 25(21): 26253-26261. DOI: 10.1364 / OE.25.026253), which is not conducive to promotion and application. Summary of the invention

[0008] The object of the present invention is to provide a compact dual-wavelength common-path holographic microscope, in which reference light and object light, two-wavelength light beams are separated and angle-adjusted by simple optical elements and adjustment methods, and the arrangement is compact and real-time fast.

[0009] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0010] A compact dual-wavelength common-path holographic microscope, comprising a first laser light source with a wavelength of λ1, a second laser light source with a wavelength of λ2, a first collimating beam expansion system, a second collimating beam expansion system, a first polarizer, a second polarizer, a first non-polarizing beam splitter prism, a second non-polarizing beam splitter prism, a plate beam splitter, a microscope objective, a polarizing beam splitter prism and an image sensor, wherein the optical path structure of the holographic microscope is:

[0011] The laser beam with a wavelength of λ1 emitted by the first laser light source is collimated and expanded by the first collimation and expansion system, and then modulated into a vertically polarized laser beam by the first polarizer;

[0012] The laser beam with a wavelength of λ2 emitted by the second laser light source is collimated and expanded by the second collimation and expansion system, and then modulated into a horizontally polarized laser beam by the second polarizer;

[0013] The vertically polarized laser beam and the horizontally polarized laser beam are combined by a first non-polarized beam splitter prism and a second non-polarized beam splitter prism, and are separated into an object beam and a reference beam by a plate beam splitter.

[0014] The object light is the transmitted light passing through the plate beam splitter, and irradiates the sample through the microscope objective lens; the reference light is the reflected light passing through the plate beam splitter; the laser beams at two wavelengths are separated after passing through the polarization beam splitter prism, and the reference light and object light at each wavelength interfere with each other respectively, and the holographic image is recorded by the image sensor.

[0015] The laser light source is used to emit a continuous laser light beam as a detection light source.

[0016] As an improvement of the present invention, the first laser light source and the second laser light source are single longitudinal mode lasers.

[0017] The composite wavelength of the two wavelengths is greater than the optical length of the sample to be measured.

[0018] The first non-polarizing beam splitter prism and the second non-polarizing beam splitter prism are respectively placed at 45° to the optical axis. The selection of the 45° angle can separate the two wavelengths of light, and the propagation direction of the light remains unchanged after passing through the polarizing beam splitter prism.

[0019] The splitting ratios of the first non-polarizing beam splitter prism and the second non-polarizing beam splitter prism are both 50:50 [R:T], so that the intensity of the light beams of the two wavelengths remains consistent.

[0020] As an improvement of the present invention, the plate-type beam splitter is mounted on a translation stage for angle fine-tuning to control the off-axis angle; in order to match the beam intensities of the object light and the reference light and enhance the interference effect, the splitting ratio of the plate-type beam splitter is set to 30:70 [R:T], and the splitting ratio of the polarization beam splitter prism is set to 50:50 [R:T].

[0021] As an improvement of the present invention, the holographic microscope includes a sample stage for placing a sample, and the sample stage is placed on the object focal plane of the microscope objective.

[0022] The holographic microscope includes a computer processing system for controlling an image sensor and processing a holographic image received by the image sensor, wherein the holographic image includes two holograms at two wavelengths of λ1 and λ2, and the processing method is as follows:

[0023] The same sample area of ​​the two holograms is extracted by feature matching algorithm and numerically reconstructed to obtain the wrapped phase distribution at two wavelengths.

[0024] After unwrapping the wrapped phase using the phase unwrapping algorithm, the system distortion contained in the phase distribution is removed using the phase distortion numerical compensation algorithm;

[0025] After obtaining the wrapped phase at two wavelengths, the accurate phase distribution is solved using a dual-wavelength phase recovery algorithm, and finally the sample surface height is recovered using the linear relationship between the phase and the sample surface height.

[0026] As an improvement of the present invention, the phase unwrapping algorithm is a branch-cutting phase unwrapping algorithm based on path tracking, which identifies positive and negative residual points and connects adjacent residual points to form an optimal branch cut line, and determines an integral path that does not pass through this branch cut line.

[0027] Among them, system distortion usually consists of the deviation of the optical axis alignment of the optical element, the introduction of the off-axis angle and the tilt distortion caused by the deviation of the camera target surface, the surface distortion caused by the microscope objective and a small amount of high-order distortion.

[0028] As an improvement of the present invention, the phase distortion numerical compensation algorithm is a ZPF compensation method, which accurately fits and removes the overall distortion of the system by matching the coefficients in the Zernike inequality.

[0029] As an improvement of the present invention, the dual-wavelength phase recovery algorithm is a dual-wavelength phase unwrapping algorithm based on linear programming.

[0030] Compared with the prior art, the dual-wavelength common-path holographic microscope provided by the present invention has the following advantages:

[0031] 1. The present invention has the characteristics of in-situ, real-time and fast holographic technology, and has the advantages of high vibration resistance and high time stability of common path optical path arrangement.

[0032] 2. In the present invention, the separation and adjustment of the light beam do not require complicated optical elements and adjustment steps. The reference light and the object light, the light beams of two wavelengths, can be separated and the angles adjusted by simple optical elements and adjustment methods.

[0033] 3. The optical path arrangement of the present invention is compact and easy to promote and apply; it has obvious advantages in the morphological characterization of precise structures and high-precision dynamic imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a schematic structural diagram of a compact dual-wavelength common-path holographic microscope in an embodiment of the present invention;

[0035] Numbers in the figure: 1. First laser light source; 2. Second laser light source; 3. First collimating beam expanding system; 4. Second collimating beam expanding system; 5. First polarizer; 6. Second polarizer; 7. First non-polarizing beam splitter prism; 8. Second non-polarizing beam splitter prism; 9. Plate beam splitter; 10. Microscope objective; 11. Sample stage; 12. Polarizing beam splitter prism; 13. Image sensor; 14. Computer processing system. DETAILED DESCRIPTION

[0036] The specific implementation methods of the present invention are described in detail with reference to the accompanying drawings and embodiments. The described embodiments are only part of the embodiments of the present invention and are not intended to be all limitations of the present invention. Based on the embodiments of the present invention, all other embodiments made by ordinary technicians in the field without creative work are within the protection scope of the present invention.

[0037] like Figure 1 As shown, the compact dual-wavelength common-path holographic microscope provided in this embodiment includes a laser light source, a collimating beam expansion system, a polarizer, a non-polarizing beam splitter, a plate beam splitter 9, a microscope objective 10, a sample stage 11, a polarizing beam splitter 12, an image sensor 13, and a computer processing system 14. The laser light source includes a first laser light source 1 and a second laser light source 2, the collimating beam expansion system includes a first collimating beam expansion system 3 and a second collimating beam expansion system 4, the polarizer includes a first polarizer 5 and a second polarizer 6, the non-polarizing beam splitter prism includes a first non-polarizing beam splitter prism 7 and a second non-polarizing beam splitter prism 8, the second non-polarizing beam splitter prism 8, the plate beam splitter 9, and the microscope objective 10 are coaxially placed; the laser light source 1 and the laser light source 2 are used to emit continuous laser light; the image sensor 13 is used to record the off-axis holographic image generated by the interference of the reference light and the object light at two wavelengths; the computer processing system 14 is used to control the image sensor 13 and process the off-axis holographic image received by the image sensor 13.

[0038] It should be noted that the laser light source used in this embodiment is a single longitudinal mode laser, which has the characteristics of high-performance continuous and ultra-low noise, and is suitable for holographic microscopic imaging. The sample stage 11 is placed on the object focal plane of the microscope objective 10; the plate beam splitter 9 is installed on a two-dimensional micro-displacement stage to fine-tune the angle to control the off-axis angle; the beam splitter 9 has a beam splitting ratio of 30:70 [R:T]; the first non-polarizing beam splitter prism 7 and the second non-polarizing beam splitter prism 8 have a beam splitting ratio of 50:50 [R:T].

[0039] The specific process of the computer processing system 14 processing the off-axis holographic image is as follows:

[0040] The image sensor 13 records the off-axis holographic images at two wavelengths, and obtains the wrapped phase matrix of the object on the object plane through numerical reconstruction; then the wrapped phase matrices at two single wavelengths are obtained through the phase recovery algorithm, including the phase unwrapping algorithm and the distortion numerical compensation algorithm; the surface height of the sample to be tested at a single wavelength is obtained through the linear relationship between the surface height and the phase distribution; the accurate height distribution of the sample to be tested is obtained through the dual-wavelength phase recovery algorithm; and the results are saved and output. Specifically:

[0041] (1) Recording and numerical reconstruction of single-wavelength off-axis holographic images:

[0042] When recording and numerically reconstructing the above off-axis holographic image, it is assumed that the two beams (λ2>λ1) respectively illuminate the sample to be tested and are recorded by the image sensor to form off-axis holographic images at two wavelengths. The synthetic wavelength of the system The object light beam and the reference light beam reflected by the sample to be measured interfere and propagate to the image sensor recording plane. The complex amplitude and intensity distribution of the total light field on the image sensor recording plane can be expressed as follows:

[0043]

[0044] Among them, O(x, y) and R(x, y) are the complex amplitudes of the object light and the reference light, respectively, and α and β are the off-axis angles of the two light beams k1 and k2 in the off-axis holographic optical path setting.

[0045] When numerical reconstruction is performed using the angular spectrum method, the reference beam is used to reproduce the object light field. The complex amplitude distribution of the object light field on the object plane can be expressed as:

[0046] O1'(x,y)=IFFT{FFT[R1(x,y)I1(x,y)]G V1 (f x ,f y )}

[0047] O2'(x,y)=IFFT{FFT[R2(x,y)I2(x,y)]GV2 (f x ,f y )}

[0048] Among them, IFFT stands for inverse Fourier transform, FFT stands for Fourier transform, G V1 and G V2 represents the transfer function of the angular spectrum diffraction at two wavelengths, f x and f y Represents the frequency of the light wave in the x and y directions.

[0049] After obtaining the complex amplitude distribution of the object light field, its intensity and phase distribution can be obtained by the following formula:

[0050] I i (x,y)=|O i '(x,y)| 2

[0051]

[0052] (2) Phase unwrapping and distortion numerical compensation of single wavelength phase distribution:

[0053] The phase distribution obtained by the above calculation process It contains not only the phase of the sample to be tested, but also the distortion in the system. Therefore, the phase distribution is wrapped between [-π,π]. The phase recovery of a single wavelength is performed by the branch cutting phase unwrapping algorithm and the distortion numerical compensation method. The overall phase difference of the system is removed by the numerical compensation method of ZPF polynomial fitting:

[0054]

[0055] Among them, angle represents the representation of phase, is the distortion of the system, is the wrapped phase with system distortion removed.

[0056] (3) Calculate the surface height distribution of the sample under two single wavelengths:

[0057] After obtaining the wrapped phase of a single wavelength, it is necessary to solve the height distribution of the single wavelength through the linear relationship between the surface height and the phase. When the illumination direction and the observation direction are both perpendicular to the zero phase plane, the relationship between the object height and the phase can be simplified as:

[0058]

[0059] (4) Dual-wavelength accurate phase recovery:

[0060] It should be noted that obtaining the accurate phase by the single-wavelength phase subtraction method will amplify the noise in the single-wavelength result, which is not conducive to the morphological characterization of the precise structure. The dual-wavelength phase recovery algorithm in the present invention adopts a method based on linear programming. The specific execution method is:

[0061] The height distribution obtained from two single-wavelength phase images can usually be expressed as:

[0062]

[0063] Among them, m1 and m2 are unknown non-negative integers at two wavelengths at the pixel point (x, y). Next, the minimum square value of the height difference is applied to search for the best integer matching pair of m1 and m2:

[0064]

[0065] After obtaining the best integer matching pair of m1 and m2, substitute it into the height solution formula of any wavelength to solve the accurate height result of the sample to be measured.

[0066] (5) Result output: The computer processing system saves and outputs the measurement results in the form of three-dimensional distribution data.

[0067] While embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the invention, and the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A compact dual-wavelength common-path holographic microscope, characterized in that: The holographic microscope comprises a first laser light source (1) with a wavelength of λ1, a second laser light source (2) with a wavelength of λ2, a first collimating beam expanding system (3), a second collimating beam expanding system (4), a first polarizing plate (5), a second polarizing plate (6), a first non-polarizing beam splitting prism (7), a second non-polarizing beam splitting prism (8), a plate beam splitter (9), a microscope objective lens (10), a polarizing beam splitting prism (12) and an image sensor (13). The optical path structure of the holographic microscope is as follows: A first laser light source (1) emits a laser beam with a wavelength of λ1, which is collimated and expanded by a first collimating and expanding system (3), and then modulated into a vertically polarized laser beam by a first polarizer (5); The second laser light source (2) emits a laser beam with a wavelength of λ2, which is collimated and expanded by a second collimating and expanding system (4), and then modulated into a horizontally polarized laser beam by a second polarizer (6); The vertically polarized laser beam and the horizontally polarized laser beam are combined through a first non-polarized beam splitting prism (7) and a second non-polarized beam splitting prism (8), and are separated into object light and reference light through a plate-type beam splitter (9); The object light is the transmitted light passing through the plate-type beam splitter (9), and irradiates the sample through the microscope objective lens (10); the reference light is the reflected light passing through the plate-type beam splitter (9); the laser beams at two wavelengths are separated after passing through the polarization beam splitter prism (12), the reference light and the object light at each wavelength interfere with each other, and the image sensor (13) records the holographic image.

2. The compact dual-wavelength common-path holographic microscope according to claim 1, characterized in that: The first laser light source (1) and the second laser light source (2) are single longitudinal mode lasers.

3. The compact dual-wavelength common-path holographic microscope according to claim 1, characterized in that: The composite wavelength of the two wavelengths is greater than the optical length of the sample to be measured.

4. The compact dual-wavelength common-path holographic microscope according to claim 1, characterized in that: The first non-polarizing beam splitter prism (7) and the second non-polarizing beam splitter prism (8) are respectively placed at an angle of 45° to the optical axis, and the splitting ratio of the first non-polarizing beam splitter prism (7) and the second non-polarizing beam splitter prism (8) is 50:50 [R:T].

5. The compact dual-wavelength common-path holographic microscope according to claim 1, characterized in that: The plate-type beam splitter (9) is mounted on a displacement platform for fine-angle adjustment to control the off-axis angle, and the splitting ratio of the plate-type beam splitter (9) is 30:70 [R:T]; the splitting ratio of the polarization beam splitter prism (12) is 50:50 [R:T].

6. The compact dual-wavelength common-path holographic microscope according to claim 1, characterized in that: The holographic microscope comprises a sample stage (11) for placing a sample, and the sample stage (11) is placed on the object focal plane of the microscope objective lens (10).

7. The compact dual-wavelength common-path holographic microscope according to any one of claims 1 to 6, characterized in that: The holographic microscope comprises a computer processing system (14) for controlling an image sensor (13) and processing a holographic image received by the image sensor (13), wherein the holographic image comprises two holograms at two wavelengths of λ1 and λ2, and the processing method is as follows: The same sample area of ​​the two holograms is extracted by feature matching algorithm and numerically reconstructed to obtain the wrapped phase distribution at two wavelengths. After unwrapping the wrapped phase using the phase unwrapping algorithm, the system distortion contained in the phase distribution is removed using the phase distortion numerical compensation algorithm; After obtaining the wrapped phase at two wavelengths, the accurate phase distribution is solved using a dual-wavelength phase recovery algorithm, and finally the sample surface height is recovered using the linear relationship between the phase and the sample surface height.

8. The compact dual-wavelength common-path holographic microscope according to claim 7, characterized in that: The phase unwrapping algorithm is a branch-cutting phase unwrapping algorithm based on path tracking, which forms an optimal branch cut line by identifying positive and negative residual points and connecting adjacent residual points, and determines an integral path that does not pass through this branch cut line.

9. The compact dual-wavelength common-path holographic microscope according to claim 7, characterized in that: The phase distortion numerical compensation algorithm is a ZPF compensation method, which accurately fits and removes the overall distortion of the system by matching the coefficients in the Zernike inequality.

10. The compact dual-wavelength common-path holographic microscope according to claim 7, characterized in that: The dual-wavelength phase recovery algorithm is a dual-wavelength phase unwrapping algorithm based on linear programming.

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