CN113960906B - Point diffraction digital holographic microscopic device based on multimode optical fiber - Google Patents
Point diffraction digital holographic microscopic device based on multimode optical fiber Download PDFInfo
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- G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
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Abstract
The invention discloses a point diffraction digital holographic microscopy device based on multimode fiber illumination, which comprises a partially coherent light generation module, a telescope system, a reference light separation module and an image acquisition module, wherein the partially coherent light generation module is used for generating partially coherent illumination light and comprises a laser, a first microscope objective, a ground glass sheet, an imaging unit and a multimode fiber unit, wherein the first microscope objective, the ground glass sheet, the imaging unit and the multimode fiber unit are sequentially arranged along the light path of the laser; the telescope system is used for obtaining object light wave field distribution with sample information; the object reference light separation module is used for carrying out diffraction light separation on object light waves with sample information from the telescope system to generate object light and reference light with orthogonal polarization directions; the image acquisition module is used for acquiring the hologram generated by the object light and the reference light. The microscopic device has the advantages of real-time amplitude/phase quantitative microscopic imaging, good vibration resistance, high signal-to-noise ratio, simple structure and the like.
Description
Technical Field
The invention belongs to the technical field of microscopic imaging, and particularly relates to a point diffraction digital holographic microscopic device based on multimode optical fibers, which can be used for measuring the three-dimensional appearance or refractive index distribution of a tiny object.
Background
Digital Holographic Microscopy (DHM), as a quantitative phase imaging technique, combines the Digital Holographic technique with the optical Microscopy technique, and can quantitatively obtain information such as three-dimensional morphology, refractive index distribution and the like of samples such as cells and the like from intensity and phase images obtained by reconstructing a hologram, thereby being an effective three-dimensional imaging technique with full-field quantification, no damage, no contact, rapidness and high resolution. DHM is currently widely used in the fields of industrial detection, biomedical imaging, special beam generation, gas fluid visualization, and adaptive imaging.
Although digital holographic microscopy has advantages not comparable to conventional optical microscopy, it still has some disadvantages and challenges. The existing DHM devices mostly adopt an optical path structure with separated object and reference lights, that is, the object light and the reference light interfere with each other after propagating for a certain distance along different paths, so as to form a hologram, and thus, the disturbance of the external environment can cause different influences on the object light and the reference light, so that the hologram is very susceptible to the influence of the environmental disturbance. Therefore, how to improve the stability of the device becomes an unavoidable problem in the practical use of DHM. Common methods for improving the stability of the device include an air cushion vibration isolation method, a vacuum closed vibration isolation method and a negative feedback electronic circuit vibration isolation method.
Popescu et al improve the stability of the system by adding a feedback system to the device, which, although effective in isolating vibration, is expensive and adds to the structural complexity of the device. In addition, the influence of environmental disturbance on phase imaging can be overcome by using two optical paths of the objective-parameter common path and the single-beam phase imaging: (1) the object-parameter common path can improve the stability of the device, the object light and the reference light reach the surface of the detector through the same path in the imaging process and generate interference patterns, and the optical path difference between the object light and the reference light cannot be influenced because the environment disturbance has the same influence on the object light and the reference light. (2) The single-beam phase imaging can improve the stability of the device, and the imaging method realizes phase imaging by recording the hologram of the object light, so that the anti-interference capability of the device is enhanced while the experimental light path is simplified. At present, the objective-parameter common-path interference microscopy mainly comprises the following steps: fizeau interference microscopy, differential interference microscopy, phase contrast interference microscopy, and the like. In recent years, the unit such as Popescu et al and Western Anlunche of Chinese academy of sciences in China put forward the objective-parameter common-path point diffraction digital holographic microscopy, and effectively reduces the influence of environmental disturbance on quantitative phase imaging. The existing point diffraction phase microscopy technology forms reference light by filtering object light waves, so that the light intensity of the reference light is related to a detected sample, and the interference patterns of all samples cannot be guaranteed to have high fringe contrast. On the basis, the Sigan electronic science and technology university provides a point diffraction digital holographic microscopic light path based on a polarization grating, and the contrast of a hologram can be adjusted by adjusting the polarization state of incident light, so that DHM imaging with high signal-to-noise ratio is ensured.
In addition, conventional DHMs generally use laser as an illumination light source, and the recorded hologram has high coherent noise including speckle noise and parasitic interference fringes, which inevitably affects the quality of phase imaging, thereby reducing the sensitivity of phase measurement. To improve the sensitivity of phase measurements, DHM based on partially coherent light illumination has emerged and is increasingly attracting widespread attention. When the partially coherent light source is used for illumination, coherent noise can be effectively reduced, and the measurement precision is improved. At present, the spatial phase noise during white light illumination can reach 0.6nm, and is reduced by one order of magnitude compared with the noise during laser illumination. At present, an objective parameter common path point diffraction digital holographic microscope based on LED illumination is provided, and low-noise and high-stability quantitative phase imaging can be realized. However, LEDs are quasi-spread light sources, the size of the light emitting point of which is in the order of millimeters, causing aliasing of the object light spectrum. When the object spectrum is filtered by using the pinhole to generate the reference light without containing the sample information, the sample information is contained in the reference light due to the overlarge pinhole, and the reference light is low in intensity and low in fringe contrast when the pinhole is too small.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a point diffraction digital holographic microscopy device based on multimode optical fibers. The technical problem to be solved by the invention is realized by the following technical scheme:
the invention provides a point diffraction digital holographic microscopic device based on multimode fiber illumination, which comprises a partially coherent light generation module, a telescope system, an object reference light separation module and an image acquisition module which are sequentially arranged along the direction of a light path, wherein,
the partial coherent light generation module is used for generating partial coherent illumination light and comprises a laser, a first microscope objective, a ground glass sheet, an imaging unit and a multi-mode optical fiber unit, wherein the first microscope objective, the ground glass sheet, the imaging unit and the multi-mode optical fiber unit are sequentially arranged along a light path of the laser;
the telescope system is used for acquiring a scattering signal of a sample by using the partially coherent illumination light and amplifying the scattering signal to obtain light field distribution with sample information;
the object reference light separation module is used for diffracting light with sample information from the telescope system to +/-1 order, wherein the +/-1 order diffracted light is respectively left-handed circularly polarized light and right-handed circularly polarized light with orthogonal polarization directions and is used as object light and reference light;
the image acquisition module is used for acquiring the hologram generated by the object light and the reference light.
In one embodiment of the present invention, the module for generating partially coherent light further comprises a motor connected to the ground glass sheet for adjusting the rotation speed of the ground glass sheet.
In one embodiment of the invention, the ground glass sheet is ground on one side, and the ground side is close to the focus of the first microscope objective.
In one embodiment of the present invention, the imaging unit includes a first thin lens and a second thin lens sequentially disposed along an optical path.
In one embodiment of the present invention, the multimode optical fiber unit includes a first optical fiber head, a second optical fiber head, and a multimode optical fiber connected between the first optical fiber head and the second optical fiber head, wherein light from the second thin lens is incident from the first optical fiber head and is emitted from the second optical fiber head.
In one embodiment of the present invention, the partially coherent light generation module further comprises a third thin lens, an adjustable first polarizer and a quarter wave plate, which are sequentially disposed in the emitting direction of the second optical fiber head, wherein the second optical fiber head is located at the focal point of the third thin lens;
the first polarizer and the quarter-wave plate are used for adjusting the relative light intensity of the object light and the reference light so as to realize the maximization of the holographic stripe contrast.
In one embodiment of the invention, the telescopic system comprises a second microscope objective and a fourth thin lens arranged in sequence along the optical path, wherein the sample is placed at the front focal plane of the second microscope objective.
In one embodiment of the present invention, the object reference light separation module includes a polarization grating, a fifth thin lens, a pinhole filter, a sixth thin lens, and a second polarizer, which are sequentially disposed along an optical path, wherein,
the polarization grating is positioned at the front focal plane of the fifth thin lens and is used for diffracting the light with the sample information from the telescope system to the +/-1 order direction to form +1 order diffraction light and-1 order diffraction light;
the pinhole filter is positioned at the back focal plane of the fifth thin lens and comprises a large hole and a pinhole, the large hole is used for enabling the +1 st order diffraction light to pass through so as to generate object light containing sample information, and the pinhole is used for low-pass filtering the-1 st order diffraction light so as to generate reference light without sample information;
the second polarizer is used for polarizing the object light and the reference light so that the object light and the reference light have the same polarization direction.
In one embodiment of the invention, the core diameter D of the multimode optical fiber fiber And the diameter D of the pinhole on the pinhole filter PH Satisfies the following relationship:
D PH ＝M×D fiber ，
wherein M represents the total magnification between the second fiber tip and the pinhole filter.
In one embodiment of the invention, a first plane mirror is arranged between the laser and the first microscope objective, and a second plane mirror is arranged between the fourth thin lens and the polarization grating.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention relates to a point diffraction digital holographic microscopic device based on multimode optical fibers, which utilizes laser as an illumination light source, a rotating ground glass sheet is placed before the laser is coupled into the multimode optical fibers, so that the laser forms partial coherent light, the size of a luminous point of the partial coherent light is strictly controlled to be the core diameter of the multimode optical fibers through the multimode optical fibers, and the device is very suitable for pinhole filtering in the point diffraction digital holographic microscopic device to generate ideal reference light. The rotating ground glass sheet is adopted to obtain partial coherent light, the background noise of the coherent light can be inhibited, the imaging signal-to-noise ratio is high, the multimode optical fiber is used for receiving and transmitting dynamic scattered light, the size of an optical fiber core (as an actual light emitting point) and the size of a pinhole filter are perfectly compatible, and finally, an ideal reference light and a hologram with high fringe contrast can be formed.
2. Since the object light and the reference light pass through the same optical elements, the point-diffraction digital holographic microscopy device of the invention has very good immunity to environmental disturbances.
3. The point diffraction digital holographic microscopy device utilizes the polarization characteristic of the polarization grating, adjusts the image contrast by rotating the first polaroid or the quarter-wave plate, and overcomes the defect of low contrast of the traditional point diffraction phase microscopic stripe. The point diffraction digital holographic microscopic device has the advantages of high stability, real-time amplitude/phase imaging and the like, and can be widely applied to multiple fields of biomedical imaging, industrial detection and the like.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a schematic structural diagram of a multimode fiber-based point diffraction digital holographic microscopy device provided by an embodiment of the invention;
FIG. 2 is a schematic diagram of the size matching of an optical fiber core and a pinhole filter according to an embodiment of the present invention;
FIG. 3 is a graph of comparative results obtained using a prior art imaging device and a point diffraction digital holographic microscopy device according to an embodiment of the present invention;
FIG. 4 is a graph showing the imaging results obtained by using the point diffraction digital holographic microscopy apparatus provided by the embodiment of the present invention;
fig. 5 is a photographic reconstruction result of COS7 cells obtained by using the point diffraction digital holographic microscopy apparatus provided by the embodiment of the present invention, wherein fig. 5(a) is an off-axis hologram of a sample, fig. 5(b) is a hologram spectrum, fig. 5(c) is a reconstructed amplitude image, and fig. 5(d) is a reconstructed phase image.
Detailed Description
To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, a point diffraction digital holographic microscope device based on multimode fiber illumination according to the present invention will be described in detail with reference to the accompanying drawings and the detailed description.
The foregoing and other technical matters, features and effects of the present invention will be apparent from the following detailed description of the embodiments, which is to be read in connection with the accompanying drawings. The technical means and effects of the present invention adopted to achieve the predetermined purpose can be more deeply and specifically understood through the description of the specific embodiments, however, the attached drawings are provided for reference and description only and are not used for limiting the technical scheme of the present invention.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that an article or device that comprises a list of elements does not include only those elements but may include other elements not expressly listed. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of additional like elements in the article or device comprising the element.
Referring to fig. 1, fig. 1 is a schematic structural diagram of a multimode fiber-based point diffraction digital holographic microscopy device according to an embodiment of the present invention. The point diffraction digital holographic microscope device comprises a partially coherent light generation module, a telescope system, an object reference light separation module and an image acquisition module which are sequentially arranged along the direction of a light path. The partially coherent light generation module is used for generating partially coherent illumination light and comprises a laser 1, a first microscope objective 3, a ground glass sheet 4, an imaging unit and a multimode optical fiber unit, wherein the first microscope objective 3, the ground glass sheet 4, the imaging unit and the multimode optical fiber unit are sequentially arranged along a light path of the laser 1; the telescope system is used for acquiring a scattering signal of a sample by using the partially coherent illumination light and amplifying the scattering signal to obtain light field distribution with sample information; the object reference light separation module is used for diffracting light with sample information from the telescope system to +/-1 order, wherein the +/-1 order diffracted light is respectively left-handed circularly polarized light and right-handed circularly polarized light with orthogonal polarization directions and is used as object light and reference light; the image acquisition module is used for acquiring the hologram generated by the object light and the reference light.
The partially coherent light generation module also comprises a motor, the motor is connected to the ground glass sheet 4, and the rotating speed of the ground glass sheet 4 can be adjusted by applying different voltages to the motor. The frosted glass sheet 4 of the present embodiment is polished on a single surface, and the polished surface is close to the focus of the first microscope objective 3, so as to improve the collection efficiency of the multimode optical fiber unit on the partially coherent light. By adjusting the distance between the polished surface and the focal point of the first microscope objective 3, the coherence properties of the partially coherent light can be adjusted. When the ground surface of the ground glass plate 4 is away from the focal point of the first microscope objective 3, the spatial coherence of the illumination light gradually decreases. Preferably, the rotation speed of the ground glass sheet 4 is 30 to 60 revolutions per second.
The imaging unit includes a first thin lens 5 and a second thin lens 6 which are sequentially arranged along an optical path. The multimode optical fiber unit includes a first optical fiber head 7, a second optical fiber head 9, and a multimode optical fiber 8 connected between the first optical fiber head 7 and the second optical fiber head 9, wherein light from the second thin lens 6 is incident from the first optical fiber head 7 and is emitted from the second optical fiber head 9. The multimode optical fiber 8 is used to define the diameter size of the light. In this embodiment, the partially coherent illumination of the point diffraction digital holographic microscope requires that the effective light emitting point of the partially coherent light is 50-200 μm and the divergence angle is 5-20, so that the core diameter of the multimode optical fiber 8 is about 50-200 μm. The first optical fiber stub 7 and the second optical fiber stub 9 are flanges or other supports that are directly connected to the multimode optical fiber 8.
The laser light emitted from the laser 1 passes through the ground glass sheet 4 rotating at a high speed, and the coherence of the light is broken to generate partially coherent light. The partially coherent light is coupled into a first fiber head 7 by an imaging system consisting of a first thin lens 5 and a second thin lens 6, passes through a multimode fiber 8, and is guided out through a second fiber head 9.
In the present embodiment, the laser wavelength emitted by the laser 1 is in the visible light range, the output laser power is stable, and the laser has an appropriate coherence length. Preferably, the laser 1 is a solid laser, the diameter of the emergent light spot is 4mm, the wavelength range is 532nm +/-5 nm (full width at half maximum 10nm), the linear polarization is carried out, and the polarization direction is the horizontal direction. The magnification of the first microscope objective lens 3 is 20X, the first thin lens 5 and the second thin lens 6 are both achromatic double-cemented lenses, and the focal length of the first thin lens 5 is f 1 75mm, the focal length of the second thin lens 6 is f 2 ＝75mm。
Further, the partially coherent light generating module further comprises a third thin lens 10, an adjustable first polarizer 11 and a quarter-wave plate 12, which are sequentially arranged in the emitting direction of the second optical fiber head 9, wherein the second optical fiber head 9 is located at the focal point of the third thin lens 10, and the first polarizer 11 and the quarter-wave plate 12 are used for adjusting the relative light intensities of the object light and the reference light, so as to maximize the fringe contrast of the hologram. By adjusting the first polarizer 11 and the quarter-wave plate 12, the light intensity reaching the image acquisition module can be brought to a near saturation state. Preferably, the image acquisition module is a black and white CCD camera with proper gray scale, pixel size and pixel number, and the focal length of the third thin lens 10 is f 3 ＝12mm。
In this embodiment, the telescope system includes a second micro objective 14 and a fourth thin lens 15 sequentially disposed along an optical path, where the sample 13 is placed at a front focal plane of the second micro objective 14, the second micro objective 14 is used for expanding the beam of the sample 13 to achieve microscopic imaging, and the axial position of the sample 13 is adjusted to enable the CCD camera 22 to have a compact imageA clear image of the sample appeared. Preferably, the magnification of the second microscope objective 14 is 10X, and the numerical aperture NA is 0.45. The fourth thin lens 15 has a focal length f 4 ＝150mm。
Further, the object reference light separation module comprises a polarization grating 17, a fifth thin lens 18, a pinhole filter 19, a sixth thin lens 20 and a second polarizing plate 21 which are sequentially arranged along an optical path, wherein the polarization grating 17 is positioned at a front focal plane of the fifth thin lens 18 and is used for diffracting light with sample information from the telescope system to the +/-1 order direction to form +1 order diffracted light and-1 order diffracted light; the pinhole filter 15 is located at the back focal plane of the fifth thin lens 18 and includes a large hole for passing the +1 st order diffracted light to generate object light containing sample information and a pinhole for low-pass filtering the-1 st order diffracted light to generate reference light containing no sample information; the second polarizing plate 21 serves to polarize object light (+ 1-order diffracted light) and reference light (-1-order diffracted light) so that the object light and the reference light have the same polarization direction.
The polarization grating 17 of the present embodiment can diffract the incident light into ± 1 st order, and the ± 1 st order diffracted light is left circularly polarized light and right circularly polarized light, respectively. The polarization grating 17 has high diffraction efficiency on +/-1 order, and the light intensity ratio of +/-1 order diffraction light is more than 40%. Preferably, the period of the polarization grating 17 is Λ ═ 6.3 μm, since (1/Λ ═ 1/6.3 ═ 0.16 μm -1 )＞(2ν max ＝2/(0.61λ/ΝΑ)/M＝0.12μm -1 The device can separate the frequency spectrums of different diffraction light on the premise of reserving the maximum resolution of the objective lens so as to independently filter the +1 st order diffraction light, wherein M is the magnification of the objective lens, v max Representing the maximum spatial frequency of the entire device.
The polarization grating 17 is placed on the image plane of the sample 13 and imaged by a telescopic system consisting of a fifth thin lens 18 and a sixth thin lens 20 onto a CCD camera 22. At this time, the polarization grating 17 and the CCD camera 22 satisfy an imaging relationship, and the magnification thereof is the magnification f of the telescope system (the fifth thin lens 18 and the sixth thin lens 20) 6 /f 5 . In the present embodiment, the fifth thin lens 18 has a focal length f 5 50mm, firstThe six thin lenses 20 have a focal length f 6 ＝50mm。
The pinhole filter 19 may be a metal sheet with a pinhole of suitable diameter for low pass filtering the-1 st order diffracted light to ensure that the intensity of the filtered beam is approximately uniform across the field of view of the CCD camera and a large hole for passing all the +1 st order diffracted light spectrum. In order to obtain a reference light with high light intensity and high uniformity and a hologram with high fringe contrast, please refer to fig. 2, fig. 2 is a schematic diagram illustrating the size matching between the fiber core and the pinhole filter according to an embodiment of the present invention, wherein PH represents a pinhole of the pinhole filter. In the present embodiment, the core diameter D of the multimode optical fiber 8 fiber With the diameter D of the pinhole on the pinhole filter 19 PH Satisfies the following relationship:
D PH ＝M×D fiber ，
where M denotes the total amplification between the second fibre tip 9 and the pinhole filter 19. Preferably, the pinhole diameter of the pinhole filter 19 is 50 μm.
Further, a first plane reflector 2 is arranged between the laser 1 and the first microscope objective 3, a second plane reflector 16 is arranged between the fourth thin lens 15 and the polarization grating 17, and the first plane reflector 2 and the second plane reflector 16 are used for realizing the turning of the light path so as to reduce the volume of the whole device. In addition, the deflection light path also has the advantage of an inverted microscope, which is convenient for observing adherent cells in the culture dish from below.
In a specific imaging process, laser light emitted by a laser 1 is focused on ground glass 4 rotating at a high speed through a first microscope objective 3 to form partially coherent light, the partially coherent light is imaged on a first optical fiber head 7 through a first thin lens 5 and a second thin lens 6 and is guided into a multimode optical fiber 8, and the partially coherent light guided out of a second optical fiber head 9 through the multimode optical fiber 8 is used as illumination light and is focused on a sample 13 through a third thin lens 10. The sample to be measured 13 is placed on the front focal plane of a telescopic system consisting of a second microscope objective 14 and a fourth thin lens 15, and a magnified real image will appear on the back focal plane of the telescopic system. After the light path passes through the sample, an object light wave is formed, which is imaged by the telescope system onto the polarization grating 17.
The polarization grating 17 is placed on the image plane of the telescope system, and splits the light with sample information into two identical light waves propagating in the ± 1 diffraction order directions by diffraction. The ± 1 diffracted lights are left-handed circularly polarized light and right-handed circularly polarized light, respectively, and the intensity of the ± 1 diffracted light thereof is related to the polarization direction of the incident light. The polarization grating 17 and the real image of the sample are imaged by the fifth lens 18 onto the CCD camera 21 plane.
The spectra of the two optical paths propagating along the ± 1 diffraction directions appear in the back focal plane of the fifth lens 18. Wherein the +1 st order diffracted light passes through the large aperture on the pinhole filter 19, the spectrum of which is not affected, and is used as object light; the-1 st order diffracted light is filtered by the pinhole on the pinhole filter 19 to become spherical light (no longer carrying sample information) which is used as reference light. After passing through the second polarizer 21, the object reference light has the same polarization direction and interferes. The CCD camera 22 is placed on the image plane of the polarization grating 17 via the microscopic magnification system (the fifth thin lens 18, the pinhole filter 19, the sixth thin lens 20), and receives the interference pattern of the object light and the reference light. The relative intensities of the object light and the reference light can be adjusted by rotating the quarter-wave plate 12. In summary, since the object light and the reference light pass through the same optical elements, the device has less influence on the vibration of the environment.
Further, when the incident beam a passes through the sample (forming the object light O (x, y)) and is diffracted by the polarization grating 17, the +1 st order diffracted light propagates along the +1 st order direction after being diffracted by the grating and is used as the object light, and the-1 st order diffracted light is filtered by the pinhole to form the reference light, the polarization direction of which is not changed. When the object light and the reference light pass through the polarizing plate 21 and become linearly polarized light with the same polarization direction, the object light and the reference light interfere with each other on the CCD plane.
The relative intensities of the object and reference light are related to the polarization state of the incident light (determined by the orientation of the principal axis of the 1/4 wave plate). The relative light intensity of the object light and the reference light shows an opposite trend along with the change rule of the included angle theta between the polarization direction and the horizontal direction (when the relative light intensity of the object light is increased, the relative light intensity of the reference light is reduced), on the CCD surface, the object light and the reference light interfere with each other, and the intensity distribution can be expressed as:
wherein K represents the carrier frequency quantity of interference fringes on the CCD surface,
indicating the phase difference between the object light and the reference light,
the complex amplitude of the object light after passing through the second polarizer is represented,
representing the complex amplitude of the reference light after passing through the second polarizer.
By adopting the traditional off-axis digital holographic reconstruction method, the complex amplitude of the object light can be reconstructed
Wherein d is 0 The defocusing distance is represented, namely the distance from the CCD to the image surface of the sample; r D The digital reference light is exp (-iKx), and can be determined by measuring the carrier frequency quantity K' of the fringes. IR D The method is mainly used for compensating the frequency spectrum shift caused by the included angle between the object light and the reference light. FT {. cndot } and IFT {. cndot } represent Fourier transform and inverse Fourier transform, respectively. (xi, eta) represents coordinates in the frequency domain,
the value of the window function is 1 in the selected area, and the values of the other areas are 0, so that the spectral distribution of the objective light real image is selected. Using the reproduced complex amplitude O r (x,y,d 0 ) And relation
The amplitude image | O of the sample can be obtained r (x, y) | and phase image
Finally, by
The three-dimensional shape d (x, y) and the refractive index distribution of the tested sample can also be calculated, wherein,
the phase image of the sample with respect to the illumination light is shown, d is the thickness distribution of the sample, n is the refractive index distribution of the sample, and λ is the emission center wavelength of the laser light.
The imaging performance of the multimode fiber-based point diffraction digital holographic microscopy device of the embodiment is verified through experiments. Referring to fig. 3, fig. 3 is a graph showing a comparison result between a conventional imaging device and a point diffraction digital holographic microscope device according to an embodiment of the present invention. FIGS. 3(a) and 3(b) are images obtained under coherent illumination (no rotating frosted glass) and partially coherent illumination (point diffraction digital holographic microscopy device of this example), respectively; fig. 3(c) is a normalized intensity distribution along a gray scale line in fig. 3(a) and 3(b), in which the horizontal axis represents the position coordinate, expressed in terms of the number of pixels, and the vertical axis represents the normalized light intensity value. The comparison shows that the illumination is more uniform by using the point diffraction digital holographic microscopy device. Using the formula beam contrast (BFC): BFC ═ Sigma (I) i -I avg ) 2 /N/I avg And quantitatively comparing the uniformity of the light beams in the whole field of view. I is i (x, y) represents the intensity distribution of the image, I avg Is represented by I i The average value of (x, y), and N is the total number of pixels. Here, a smaller beam contrast value indicates a more uniform beam intensity distribution. The calculation result of the coherent light illumination is 0.52, the point diffraction digital holographic microscopy device of the embodiment utilizes the rotating ground glass to generate the partial coherent light illumination, and the calculation result of the beam contrast is 0.11, namely, the intensity of the partial coherent light illumination is more uniform.
Further, a calibration sample UASF-1951 was selected for an image SNR comparison experiment, and the result of imaging with coherent illumination without ground glass is shown in FIG. 3(d), and the result of partially coherent illumination with an apparatus according to an embodiment of the present invention is shown in FIG. 3 (e). Obviously, compared with the coherent illumination imaging result without loading ground glass, the coherent noise in the partially coherent illumination result is well suppressed. Meanwhile, the light intensity distribution is extracted along the horizontal short lines of fig. 3(d) and 3(e), respectively, as shown in fig. 3 (f). As a result, it can be seen that the signal-to-noise ratio of the partially coherent illumination obtained with the point diffraction digital holographic microscopy apparatus of the present embodiment is higher than that of the coherent illumination.
Meanwhile, imaging experiments were performed by loading unrotated ground glass, and high-speed-rotation ground glass, respectively, and the experimental results are shown in fig. 4, where fig. 4(a) is the imaging result obtained when unrotated ground glass is loaded, fig. 4(b) is an enlarged region of a dotted frame in fig. 4(a), fig. 4(c) is the imaging result when unrotated ground glass is not loaded, and fig. 4(d) is the imaging result when rotating ground glass is loaded at 40 rpm. The results show that by rotating the ground glass at high speed, a dynamic scattered light field can be generated and coherent noise in the results can be significantly reduced by time averaging, compared to devices loaded with non-rotated ground glass and non-loaded ground glass.
Further, in this example, cos7-80 cells were used as the sample to be measured, and the off-axis hologram I (x, y) (fig. 5(a)) and the spectral distribution of the off-axis hologram (fig. 5(b)) of the sample were obtained, and the intensity image (fig. 5(c)) and the phase image (fig. 5(d)) corresponding to the sample were obtained by using the formula (2). Comparing fig. 5(c) and 5(d), it was readily found that for this clear sample, the phase image exhibited more detailed structure of the sample than the intensity image, e.g., the cellular structure of cos7-80 cells was visible in the phase image. Note that, here, digital refocusing is not performed during reproduction, that is, the defocus distance d in equation (2) is 0 mm. In fact, when the tested sample is out of focus during imaging, digital refocusing of the sample can also be realized by changing the value of d during reproduction.
In summary, the present patent provides a point diffraction digital holographic microscopy device based on multimode fiber, which not only has the advantages of high phase measurement precision of the traditional digital holographic microscopy, and no influence of environmental disturbance on the traditional point diffraction digital holographic microscopy, but also has the following advantages: firstly, the rotating ground glass is adopted to obtain partial coherent light, so that the background noise of the coherent light can be suppressed, and the imaging signal-to-noise ratio is high; secondly, receiving and transmitting dynamic scattered light by utilizing multimode optical fibers, wherein the fiber cores (as actual luminous points) of the optical fibers are perfectly compatible with the size of the pinhole filter, and finally, an ideal reference light and a hologram with high fringe contrast are formed; finally, the polarization characteristic of the polarization diffraction grating is utilized, the contrast of the image is adjusted by rotating the first polaroid or the quarter-wave plate, and the defect of low contrast of the traditional point diffraction phase micro-stripes is overcome. In summary, the digital holographic microscopy device has the advantages of high stability, real-time amplitude/phase imaging and the like, and can be widely applied to multiple fields of biomedical imaging, industrial detection and the like.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (7)
1. A point diffraction digital holographic microscopic device based on multimode fiber illumination is characterized by comprising a partially coherent light generation module, a telescope system, a reference light separation module and an image acquisition module which are sequentially arranged along the direction of a light path,
the partial coherent light generation module is used for generating partial coherent illumination light and comprises a laser (1), a first microscope objective (3), a ground glass sheet (4), an imaging unit and a multimode optical fiber unit, wherein the first microscope objective (3), the ground glass sheet (4), the imaging unit and the multimode optical fiber unit are sequentially arranged along the optical path of the laser (1), the ground glass sheet (4) is perpendicular to the optical axis and can rotate around the optical axis to generate dynamically scattered partial coherent light, and the multimode optical fiber unit is used for collecting the dynamically scattered partial coherent light and controlling the diameter of the partial coherent light;
the telescope system is used for acquiring a scattering signal of a sample by using the partially coherent illumination light and amplifying the scattering signal to obtain light field distribution with sample information;
the object reference light separation module is used for diffracting light with sample information from the telescope system to +/-1 order, wherein the +/-1 order diffracted light is respectively left-handed circularly polarized light and right-handed circularly polarized light with orthogonal polarization directions and is used as object light and reference light;
the image acquisition module is used for acquiring a hologram generated by the object light and the reference light;
the multimode optical fiber unit comprises a first optical fiber head (7), a second optical fiber head (9) and a multimode optical fiber (8) connected between the first optical fiber head (7) and the second optical fiber head (9), wherein light rays from the imaging unit enter from the first optical fiber head (7) and exit from the second optical fiber head (9);
the object reference light separation module comprises a polarization grating (17), a fifth thin lens (18), a pinhole filter (19), a sixth thin lens (20) and a second polaroid (21) which are arranged along a light path in sequence,
the polarization grating (17) is positioned at the front focal plane of the fifth thin lens (18) and is used for diffracting light with sample information from the telescope system to +/-1 order directions to form +1 order diffraction light and-1 order diffraction light;
the pinhole filter (19) is positioned at the back focal plane of the fifth thin lens (18) and comprises a large hole and a pinhole, the large hole is used for leading the +1 st order diffraction light to pass to generate object light containing sample information, and the pinhole is used for low-pass filtering the-1 st order diffraction light to generate reference light without sample information;
the second polarizer (21) is used for polarizing the object light and the reference light so that the object light and the reference light have the same polarization direction;
the core diameter D of the multimode optical fiber (8) fiber And the diameter D of the pinhole on the pinhole filter (19) PH Satisfies the following relationship:
D PH ＝M×D fiber ，
wherein M represents the total magnification between the second fiber head (9) and the pinhole filter (19).
2. The multimode fiber illumination-based point diffraction digital holographic microscopy device according to claim 1, wherein the partially coherent light generation module further comprises a motor connected to the ground glass sheet (4) for adjusting the rotation speed of the ground glass sheet (4).
3. The multimode fiber illumination-based point diffraction digital holographic microscopy device as claimed in claim 1, wherein the ground glass sheet (4) is ground on one side, and the ground side is close to the focus of the first microscope objective (3).
4. The multimode fiber illumination-based point diffraction digital holographic microscopy device according to claim 1, wherein the imaging unit comprises a first thin lens (5) and a second thin lens (6) arranged in sequence along the optical path.
5. The multimode fiber illumination-based point diffraction digital holographic microscopy device according to claim 4, wherein the partially coherent light generation module further comprises a third thin lens (10) arranged in the exit direction of the second fiber head (9), and an adjustable first polarizer (11) and a quarter wave plate (12), wherein the second fiber head (9) is located at the focus of the third thin lens (10); the first polarizer (11) and the quarter-wave plate (12) are used for adjusting the relative light intensity of the object light and the reference light so as to realize maximization of the holographic stripe contrast.
6. The multimode fiber illumination-based point diffraction digital holographic microscopy device according to claim 5, wherein the telescopic system comprises a second microscope objective (14) and a fourth thin lens (15) arranged in sequence along the optical path, wherein the sample (13) is placed at the front focal plane of the second microscope objective (14).
7. The multimode fiber illumination-based point diffraction digital holographic microscopy device according to claim 6, wherein a first plane mirror (2) is arranged between the laser (1) and the first microscope objective (3), and a second plane mirror (16) is arranged between the fourth thin lens (15) and the polarization grating (17).
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