A quantitative phase contrast microscopy imaging device based on partially coherent light illumination

Abstract

The present invention discloses a quantitative phase contrast microscopy imaging device based on partially coherent light illumination, comprising a partially coherent illumination module, a microscopy imaging system, a spectrum modulation module, and an image acquisition module, which are sequentially arranged along an optical path. The partially coherent illumination module is used to generate partially coherent light as illumination light; the microscopy imaging system is used to utilize the partially coherent light to acquire and amplify the scattered signal of a sample, thereby obtaining a light field distribution containing sample information; the spectrum modulation module is used to modulate the zero-frequency component and the high-frequency component of the incident light spectrum from the microscopy imaging system; and the image acquisition module is used to capture a hologram generated by the interference of the zero-frequency component and the high-frequency component modulated by the spectrum modulation module. The present invention utilizes a spatial light modulator to introduce different phase values ​​into the zero-frequency component of the object light to achieve phase shift, records the phase shift contrast pattern between the zero-frequency component and the high-frequency component of the object light, and ultimately reproduces the quantitative phase distribution of the sample.

Description

Technical Field

[0001] The present invention belongs to the field of microscopic imaging technology, and in particular relates to a quantitative phase contrast microscopic imaging device based on partially coherent light illumination, which can be used to measure the three-dimensional morphology or refractive index distribution of tiny objects. Background Art

[0002] Phase imaging has important applications in biomedicine. Because most biological samples are transparent or translucent, the imaging contrast under traditional microscopes is very low, hindering sample observation. Phase microscopy, combining phase imaging with optical microscopy, can quantitatively determine the three-dimensional morphology of microscopic objects or the refractive index distribution of transparent objects. Compared to traditional measurement methods such as scanning probe microscopy, this method offers the advantages of full-field measurement, no sample pretreatment (e.g., no fluorescent labeling, no need for conductive electrodes), and high measurement speed.

[0003] Digital Holographic Microscopy (DHM) is a type of quantitative phase imaging technology. DHM combines digital holographic technology with optical microscopy. By reconstructing the hologram, the three-dimensional morphology of samples such as cells and the refractive index distribution and other information can be quantitatively obtained from the obtained intensity and phase images. Digital holographic microscopy has the advantages of fast phase imaging speed and high phase measurement accuracy, but it still has some shortcomings and challenges. Most of the current DHM devices use an optical path structure with object parameter separation, that is, the object light and the reference light interfere after propagating along different paths for a certain distance. Therefore, disturbances in the external environment will have different effects on the object light and the reference light, making the hologram extremely susceptible to environmental disturbances.

[0004] In 1942, Danish scientist Zernike proposed another phase imaging technique—phase contrast interference microscopy. This technique transforms the phase information of the sample being measured into intensity information by delaying the zero-frequency component of the object light by π / 2. Because the intensity of the interference pattern and the phase of the object being measured are not linearly converted, traditional Zernike phase contrast imaging can only be used for qualitative observations. In phase contrast imaging, when Fourier transforming the object light using a lens with a focal length of 200 mm, the diameter (full width at half maximum) of the zero-frequency component of the object light on the spectral plane is approximately 20 to 50 μm. With the advent of spatial light modulators (SLMs), which typically have a pixel size of 4 to 8 μm, it is now possible to conveniently modulate the zero-frequency component of the object light. By introducing different phase values ​​(phase shifting) into the zero-frequency component and recording the resulting intensity image, quantitative phase measurement can also be achieved.

[0005] In phase contrast imaging, the zero-frequency and high-frequency components of an object are equivalent to the reference light and object light in optical interference. However, for different samples, the zero-frequency and high-frequency components of the object light often have different intensities, and the fringe contrast formed by the interference of the two (the contrast of the phase contrast image) is often not guaranteed. In 2012, a phase contrast interference microscopy method with adjustable contrast (fringe contrast) was proposed: a phase mask based on grating encoding was used to modulate the zero-frequency and high-frequency components of the object light. By changing the grayscale of the grating on the zero-frequency and high-frequency components, the relative intensity of the zero-frequency and high-frequency components in the phase contrast image is adjusted, thereby adjusting the contrast of the phase contrast pattern. At the same time, phase shifting operations can be performed by moving the grating laterally, realizing quantitative measurement of phase objects and quantitative measurement of the thickness or refractive index of transparent objects.

[0006] However, to date, phase contrast microscopy techniques for quantitative phase imaging have all used lasers as the illumination source. The resulting phase contrast images and reconstructed phase images contain speckle noise, which reduces the signal-to-noise ratio of phase imaging and also reduces the sensitivity of phase measurement. The use of LED illumination can effectively suppress speckle noise in phase contrast microscopy, but the area of ​​the LED's effective light-emitting point is large (on the order of millimeters), which will eventually cause aliasing of low-frequency and high-frequency components in the object light spectrum. This is because: in phase contrast imaging, the actual zero-frequency component distribution of the object light is equal to the convolution of the illumination light spectrum (the size of the actual light-emitting point before collimation) and the zero-frequency component of the sample under parallel light illumination (such as Figure 1 (As shown in the inset). Therefore, under LED illumination, the actual zero-frequency component of the object light is significantly broadened and aliased with other high-frequency components, making it impossible to independently phase-delay the zero-frequency component. Similar to traditional Zernike phase contrast imaging, LED-based phase contrast microscopy exhibits a severe "halo" phenomenon (incorrectly phase-modulating the low-frequency components of the object light). Summary of the Invention

[0007] To address the above-mentioned problems in the prior art, the present invention provides a quantitative phase contrast microscopy imaging device based on partially coherent light illumination. The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0008] The present invention provides a quantitative phase contrast microscopy imaging device based on partially coherent light illumination, comprising a partially coherent illumination module, a microscopy imaging system, a spectrum modulation module and an image acquisition module sequentially arranged along the light path direction, wherein:

[0009] The partially coherent illumination module is used to generate partially coherent light with an actual luminous point having a size equivalent to the zero-frequency component of the sample as illumination light;

[0010] The microscopic imaging system is used to obtain a scattered signal of the sample using the partially coherent light and amplify the scattered signal to obtain a light field distribution with sample information;

[0011] The spectrum modulation module is used to modulate the zero-frequency component and the high-frequency component of the incident light spectrum from the microscopic imaging system;

[0012] The image acquisition module is used to acquire a hologram generated by the interference of the zero-frequency component and the high-frequency component modulated by the spectrum modulation module.

[0013] In one embodiment of the present invention, the partially coherent illumination module includes a laser and a first plane reflector, a first microscope objective lens, a ground glass sheet, a first thin lens, a second thin lens, a multimode optical fiber unit, and a third thin lens, which are sequentially arranged along the optical path of the laser. The ground glass sheet is arranged perpendicular to the optical axis and can rotate around the optical axis to generate dynamically scattered partially coherent light. The multimode optical fiber unit is used to collect the dynamically scattered partially coherent light and control the diameter of the partially coherent light.

[0014] The outlet of the multimode optical fiber unit is located at the focus of the third thin lens.

[0015] 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 the first optical fiber head is located at the focus of the second thin lens, and the second optical fiber head is located at the focus of the third thin lens.

[0016] In one embodiment of the present invention, the microscopic imaging system includes a second microscope objective lens, a fourth lens, and a second plane reflector sequentially arranged along the optical path, wherein the sample is placed at the front focal plane of the second microscope objective lens.

[0017] In one embodiment of the present invention, the spectrum modulation module includes a fifth lens, a polarizer, a prism and a spatial light modulator, wherein:

[0018] The polarizer is perpendicular to the light path and converts the light incident on the polarizer into polarized light, and can maximize the modulation efficiency of the spatial light modulator;

[0019] The polarized light is incident on the first surface of the prism and then reflected to the spatial light modulator for spectrum modulation, and the modulated spectrum is incident on the second surface of the prism again;

[0020] The spatial light modulator is located at the rear focal plane of the fifth lens, and is used to modulate the zero-frequency component and the high-frequency component of the light with sample information from the microscopic imaging system respectively;

[0021] By loading a blazed grating-based phase mask on the spatial light modulator, not only can the zero-frequency component and the high-frequency component of the object light be measured separately, but also the phase-shift interference pattern between the zero-frequency component and the high-frequency component can be obtained.

[0022] In one embodiment of the present invention, the spatial light modulator is loaded with a phase mask based on a blazed grating, and the phase mask is formed by superimposing a blazed grating and a circular area located at the center of the blazed grating, wherein:

[0023] The circular area is used to cover and modulate the phase of the spectral components of the object light, and the mask plate outside the circular area is used to modulate the high-frequency components of the object light; the blazed grating is used to change the propagation direction of the modulated light wave, and can separate the modulated zero-frequency component or high-frequency component of the object light from other spectra; by superimposing different phases in the blazed grating within the circular area, the zero-frequency component of the object light can be phase delayed to achieve phase-shift phase contrast imaging.

[0024] In one embodiment of the present invention, the diameter of the circular area satisfies:

[0025] d FWHM ≤2λ / (ML)f 15 ,

[0026] Wherein, λ is the wavelength of the illumination light, M is the magnification of the telescope system composed of the second microscope objective lens and the fourth lens, and f 15 is the focal length of the fifth lens, L is the diameter of the imaging field of view;

[0027] The imaging diameter of the output end face of the multimode optical fiber on the spatial light modulator is smaller than the diameter of the circular area.

[0028] In one embodiment of the present invention, the image acquisition module includes a sixth lens, a beam splitter prism, a first CCD camera, a seventh lens and a second CCD camera, wherein:

[0029] The sixth lens is perpendicular to the light path direction, the reflection surface of the dichroic prism forms a certain angle with the sixth lens, the seventh lens is arranged in the light reflection direction of the dichroic prism, and the second CCD camera is located at the back focal plane of the seventh lens;

[0030] The first CCD camera is located in the light transmission direction of the beam splitter prism.

[0031] In one embodiment of the present invention, a grayscale grating image is loaded in the central circular area of ​​the spatial light modulator to obtain the zero-frequency component intensity distribution I0 of the object light on the first CCD camera; a grayscale grating image is loaded in the surrounding blazed grating area of ​​the spatial light modulator (18) to obtain the high-frequency component intensity distribution I d ;

[0032] Grayscale grating patterns with phase shifts of 0, 2π / 3, and 4π / 3 are loaded on the central circular area and the surrounding blazed grating area of ​​the spatial light modulator, respectively, to obtain interference intensity images I1, I2, and I3 of the object light on the first CCD camera.

[0033] In one embodiment of the present invention, the quantitative phase contrast microscopy imaging device based on partially coherent light illumination further includes a data processing module for obtaining the complex amplitude of the object light wave based on the zero-frequency component intensity distribution I0 of the object light and the interference intensity images I1, I2, and I3 of the object light.

[0034] Specifically, the expressions of the interference intensity images I1, I2, and I3 of the object light are obtained:

[0035]

[0036] Among them, O0, O d Represent the complex amplitudes of the zero-frequency component and the high-frequency component respectively, and i represents the imaginary part;

[0037] Further we get:

[0038]

[0039] O0 * O d Expressed as in, represents the phase difference between the zero-frequency component and the high-frequency component, then the complex amplitude of the object light wave is expressed as:

[0040]

[0041] Where I0 represents the intensity distribution of the zero-frequency component of the sample.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1. The quantitative phase contrast microscopy imaging device based on partially coherent light illumination uses laser as the illumination light source. A rotating frosted glass plate is placed before the laser is coupled into the multimode optical fiber, so that the laser forms partially coherent light. The luminous point size of the partially coherent light is strictly controlled to the core diameter of the multimode optical fiber through the multimode optical fiber, which is generally between 10 and 100 μm. The optical fiber core is the actual luminous point of the illumination light. When imaged onto the spatial light modulator, its diameter d FWHM It just satisfies the diameter d of the zero-frequency component of the object light calculated by optical diffraction theory. FWHM ≤2λ / (ML)f 15 This effectively prevents the high-frequency components of the sample from being mismodulated by the central circular region of the phase mask. This prevents aliasing of the zero-frequency and high-frequency components of the object light, and also reduces coherent noise.

[0044] 2. In order to reduce the speckle noise of phase contrast imaging and avoid the "halo" phenomenon in phase contrast imaging (ultimately improving the accuracy of phase measurement), our invention proposes a quantitative phase contrast microscopy technology with partially coherent illumination of small light-emitting surface elements (the diameter can be selected in the range of 10-100μm). This technology innovatively uses dynamic scattering and multimode optical fiber to generate partially coherent light illumination with an effective light-emitting point of 10 to 100μm (diameter). The size of the effective light-emitting point is consistent with the size of the zero-frequency component of the object light. The use of a matching circular phase mask will not affect the high-frequency component, and ultimately low-noise, distortion-free quantitative phase contrast imaging can be achieved. In this phase contrast microscopy optical path, the zero-frequency light path and the high-frequency light path pass through exactly the same optical elements, so the phase contrast microscopy device of the present invention has very good immunity to environmental disturbances.

[0045] 3. The quantitative phase contrast microscopy device of the present invention utilizes a spatial light modulator (SLM) loaded with a blazed grating-based mask. This allows for independent measurement of the intensity distributions of the zero-frequency and high-frequency components of the object light, as well as the recording of three phase-shifted interference patterns by introducing different phase values ​​between them. Separately measuring the intensity distributions of the zero-frequency and high-frequency components simplifies the phase reconstruction process, ultimately enabling rapid and accurate quantitative phase distribution of the sample. Furthermore, by digitally determining the size of the zero-frequency component region on the SLM, the contrast of the phase contrast imaging can be modulated.

[0046] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 1 is a schematic structural diagram of a quantitative phase contrast microscopy imaging device based on partially coherent light illumination provided by an embodiment of the present invention;

[0048] Figure 2is a grayscale pattern loaded on a spatial light modulator provided by an embodiment of the present invention;

[0049] Figure 3 is Figure 2 Intensity images or interference images obtained under different grayscale images;

[0050] Figure 4 The phase distribution diagram of the phase steps is reproduced by using the quantitative phase contrast microscopy device of the embodiment of the present invention and the existing digital holographic imaging;

[0051] Figure 5 It is the intensity distribution diagram of the zero-frequency component of the object light (the spectrum distribution of the object light when no sample is placed) in the plane of the spatial light modulator;

[0052] Figure 6 This figure compares the speckle noise in the reconstructed phase image of an optical waveguide sample illuminated by coherent light (CI) and partially coherent light (PCI).

[0053] Description of reference numerals:

[0054] 1-laser; 2-first plane mirror; 3-first microscope objective; 4-ground glass; 5-first thin lens; 6-second thin lens; 7-first optical fiber head; 8-multimode optical fiber; 9-second optical fiber head; 10-third thin lens; 11-sample; 12-second microscope objective; 13-fourth lens; 14-second plane mirror; 15-fifth lens; 16-polarizer; 17-prism; 18-spatial light modulator; 19-sixth lens; 20-beam splitter; 21-first CCD camera; 22-seventh lens; 23-second CCD camera. DETAILED DESCRIPTION

[0055] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the following is a detailed description of a quantitative phase contrast microscopy imaging device based on partially coherent light illumination proposed in accordance with the present invention, in conjunction with the accompanying drawings and specific embodiments.

[0056] The aforementioned and other technical contents, features, and effects of the present invention are clearly presented in the following detailed description of the specific embodiments in conjunction with the accompanying drawings. Through the description of the specific embodiments, a deeper and more specific understanding of the technical means and effects adopted by the present invention to achieve the intended purpose can be obtained. However, the accompanying drawings are provided for reference and illustration purposes only and are not intended to limit the technical solutions of the present invention.

[0057] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations are intended to cover non-exclusive inclusion, such that an article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of additional identical elements in the article or device comprising the element.

[0058] See Figure 1 , Figure 1 This is a schematic structural diagram of a quantitative phase contrast microscopy imaging device based on partially coherent light illumination, provided by an embodiment of the present invention. The quantitative phase contrast microscopy device includes a partially coherent illumination module, a microscopy imaging system, a spectrum modulation module, and an image acquisition module, which are sequentially arranged along the optical path. The partially coherent illumination module is used to generate partially coherent light with an actual luminous point comparable in size to the zero-frequency component of the sample as illumination light; the microscopy imaging system is used to utilize the partially coherent light to acquire and amplify the scattered signal from the sample to obtain a light field distribution containing sample information; the spectrum modulation module is used to modulate the zero-frequency component and high-frequency component of the incident light spectrum from the microscopy imaging system; and the image acquisition module is used to capture a hologram generated by the interference of the zero-frequency component and the high-frequency component modulated by the spectrum modulation module.

[0059] Furthermore, the partially coherent illumination module of this embodiment includes a laser 1 and a first plane reflector 2, a first microscope objective lens 3, a frosted glass sheet 4, a first thin lens 5 and a second thin lens 6, a multimode optical fiber unit and a third thin lens 10 arranged in sequence along the optical path of the laser. The frosted glass sheet 4 is arranged perpendicular to the optical axis and can rotate around the optical axis to generate dynamically scattered partially coherent light. The multimode optical fiber unit is used to collect the dynamically scattered partially coherent light and control the diameter of the partially coherent light; the outlet of the multimode optical fiber unit is located at the focus of the third thin lens 10.

[0060] Preferably, the multimode fiber unit includes a first fiber head 7, a second fiber head 9, and a multimode optical fiber 8 connected between the first and second fiber heads 7 and 9. The first fiber head 7 is located at the focus of the second thin lens 6, and the second fiber head 9 is located at the focus of the third thin lens 10. Light from the second thin lens 6 enters from the first fiber head 7 and exits from the second fiber head 9. The fiber diameter must match the size of the zero-frequency region.

[0061] During specific use, the light wave emitted by the laser 1 is reflected by the first plane reflector 2 and focused onto the frosted glass 4 through the first microscope objective lens 3. The light beam passes through the high-speed rotating frosted glass 4 to generate partially coherent light. The partially coherent light is imaged to the first optical fiber head 7 through the first thin lens 5 and the second thin lens 6 in sequence, and then introduced into the multimode optical fiber 8. It is output from the second optical fiber head 9 through the multimode optical fiber 8 and is used as illumination light.

[0062] Furthermore, the microscopic imaging system of this embodiment includes a second microscope objective 12, a fourth lens 13, and a second plane mirror 14, which are arranged in sequence along the optical path. The sample 11 is placed at the front focal plane of the second microscope objective 12. The function of this module is to image the spectral information of the sample onto the spatial light modulator (SLM) described below via the second microscope objective 12, the fourth lens 13, and the fifth lens 15.

[0063] The spectrum modulation module includes a fifth lens 15, a polarizer 16, a prism 17 and a spatial light modulator 18, wherein the polarizer 16 is perpendicular to the direction of the optical path and converts the light incident on the polarizer 16 into polarized light, and can make the modulation efficiency of the spatial light modulator 18 reach the maximum value; after the polarized light is incident on the first surface of the prism 17, it is reflected onto the spatial light modulator 18 for spectrum modulation, and the modulated spectrum is again incident on the second surface of the prism 17; the spatial light modulator 18 is located at the rear focal plane of the fifth lens 15, and is used to modulate the zero-frequency component and the high-frequency component of the light with sample information from the microscopic imaging system respectively; by loading a phase mask based on a blazed grating on the spatial light modulator 18, not only can the zero-frequency component and the high-frequency component of the object light be measured separately, but also the phase shift interference pattern between the zero-frequency component and the high-frequency component can be obtained.

[0064] Furthermore, the spatial light modulator 18 of this embodiment is loaded with a phase mask based on a blazed grating, wherein the phase mask is composed of a blazed grating and a circular area located at the center of the blazed grating, wherein the circular area is used to cover and modulate the phase of the spectral component of the object light, and the mask plate outside the circular area is used to modulate the high-frequency component of the object light; the blazed grating is used to change the propagation direction of the modulated light wave, and can separate the modulated zero-frequency component or high-frequency component of the object light from other spectra; by superimposing different phases in the blazed grating within the circular area, the zero-frequency component of the object light can be phase delayed to achieve phase-shift phase contrast imaging.

[0065] The image acquisition module of this embodiment includes a sixth lens 19, a beam splitter prism 20, a first CCD camera 21, a seventh lens 22, and a second CCD camera 23, wherein the sixth lens 19 is perpendicular to the light path direction, the reflective surface of the beam splitter prism 20 forms a certain angle with the sixth lens 19, the seventh lens 22 is arranged in the light reflection direction of the beam splitter prism 20, and the second CCD camera 23 is located at the back focal plane of the seventh lens 22; the first CCD camera 21 is located in the light transmission direction of the beam splitter prism 20.

[0066] Specifically, the spectrum modulation module modulates the zero-frequency component and the high-frequency component of the incident light spectrum from the imaging system respectively; the incident light passes through a polarizer 16 to form polarized light, and the polarized light is incident on the prism 17 and reflected to the spatial light modulator 18 for spectrum modulation. The modulated spectrum is incident on the prism again, undergoes Fourier transformation through the sixth lens 19, passes through the spectroscopic prism 20, and is finally imaged onto the first CCD camera 21. The first CCD camera 21 is used to collect the hologram generated by the interference of the zero-frequency component and the high-frequency component. Another light beam separated by the spectroscopic prism 20 passes through the seventh lens 22, and images the image on the spatial light modulator onto the second CCD camera 23. This light beam is used to monitor the modulation process on the spatial light modulator (SLM) in real time.

[0067] In this embodiment, a quantitative phase contrast microscopy imaging device based on partially coherent light illumination completes Zernike phase contrast imaging of a sample while suppressing coherent noise.

[0068] The illumination light generated by the partially coherent illumination module is expanded and collimated by the third thin lens 10 and then illuminates the sample 11. The sample 11 is magnified by the telescope system composed of the second microscope objective lens 12 and the fourth thin lens 13 (called object light). After being expanded and amplified, the object light is Fourier transformed by the fifth thin lens 15, and passes through the polarizer 16 and the prism 17 in sequence, and its spectrum appears on the back focal plane of the fifth thin lens 15. The spatial light modulator 18 is placed on the back focal plane of the fifth thin lens 15, and is used to adjust the object light spectrum to achieve phase contrast imaging. Specifically, the phase mask loaded on the spatial light modulator 18 is composed of a blazed grating and a circular area located at the center of the blazed grating. The blazed grating is used to change the propagation direction of the modulated light wave, thereby separating the modulated light wave from the unmodulated object light. The circular area is used to select the zero-frequency component of the object light and phase-delay the zero-frequency component of the object light to achieve phase contrast imaging.

[0069] The spectrally modulated light is then reflected back onto prism 17. After being Fourier transformed by sixth thin lens 19, the object light propagating along the +1st-order diffracted light of the blazed grating is imaged onto first CCD camera 21, eliminating the influence of zeroth-order light and other diffracted light orders not modulated by the spatial light modulator. Furthermore, along the direction of the light reflected from beamsplitter prism 20, a telescope system consisting of sixth lens 19 and seventh thin lens 22 images the pattern loaded on spatial light modulator 18, along with the object light spectrum, onto second CCD camera 23. This optical path is used to monitor the coincidence of the object light spectrum on spatial light modulator (SLM) 18 and the phase mask in real time.

[0070] It should be noted that in phase contrast imaging, the actual zero-frequency component distribution of the object light is equal to the convolution of the illumination light spectrum (the size of the actual luminous point before collimation) and the zero-frequency component of the sample under parallel light illumination, such as Figure 1 In order to avoid the high-frequency components of the sample being mismodulated by the central circular region of the phase mask, the diameter of the central circular region of the phase mask (d FWHM ) should meet the following requirements: FWHM ≤2λ / (ML)f 15 Wherein, λ is the wavelength of the illumination light, M is the magnification of the telescope system composed of the second microscope objective lens 12 and the fourth lens 13, and f 15 is the focal length of the fifth lens 15, and L is the diameter of the imaging field of view. For example, for a microscope system with M=10, the diameter of the imaging field of view is generally 0.5 mm. In this case, d is generally required to be FWHM ≤2λ / (ML)f 15 =53.2μm.

[0071] Furthermore, the diameter of the multimode optical fiber 8 needs to be such that the spectrum of the illumination light on the plane of the spatial light modulator 18 is comparable to the zero-frequency component of the object light; in other words, the imaging diameter of the output end face of the multimode optical fiber 8 on the spatial light modulator 18 is smaller than the diameter d of the circular area. FWHM .

[0072] In this embodiment, a grayscale grating image is loaded in the central circular area of ​​the spatial light modulator to obtain the zero-frequency component intensity distribution I0 of the object light on the first CCD camera; a grayscale grating image is loaded in the blazed grating area around the spatial light modulator 18 to obtain the high-frequency component intensity distribution I d ;

[0073] Grayscale grating patterns with phase shifts of 0, 2π / 3, and 4π / 3 are loaded on the central circular area and the surrounding blazed grating area of ​​the spatial light modulator, respectively, to obtain interference intensity images I1, I2, and I3 of the object light on the first CCD camera.

[0074] Specifically, the phase contrast imaging device based on partially coherent illumination can achieve quantitative phase imaging of transparent objects. Figure 2 , Figure 2 is a grayscale pattern loaded on a spatial light modulator provided by an embodiment of the present invention, wherein: Figure 2 (a) is the grayscale mask image of the zero-frequency component of the object light; Figure 2 (b) is the grayscale mask image of the high-frequency component of the object light; Figure 2 (c)- Figure 2 (e) is a grayscale mask image of an interference pattern with phase shifts of 0, 2π / 3, and 4π / 3 between the zero-frequency component and the high-frequency component of the object light. Figure 2 (a) to Figure 2 The grayscale pattern shown in (e) can be used to obtain the intensity distribution of the zero-frequency component and the high-frequency component of the object light, as well as the interference patterns with different phase shifts on the CCD surface of the first CCD camera 21. Figure 3 , Figure 3 is Figure 2 Intensity images or interference images obtained under different grayscale images, where Figure 3 (a) is the intensity image of the zero-frequency component of the object light; Figure 3 (b) is the intensity image of the high-frequency component of the object light; Figure 3 (c)-3(e) are interference patterns (phase contrast images) with phase shifts of 0, 2π / 3, and 4π / 3 between the zero-frequency and high-frequency components of the object light, respectively.

[0075] When the center grating ( Figure 2 (a)) and the surrounding grating ( Figure 2 (b)) are loaded on the spatial light modulator, and the intensity distributions of the zero-frequency component and high-frequency component of the sample, I0 and I d , respectively as Figure 3 (a) and 3(b). When there is 0( Figure 2 (c))、2π / 3( Figure 2 (d)) and 4π / 3( Figure 2 (e)) different phase shift interference patterns can be obtained, such as Figure 3 (c) to Figure 3 (e) The phase shift here refers to the different constant phase differences between the zero-frequency and high-frequency components of the light wave.

[0076] Phase shift is achieved in the zero-frequency region and the high-frequency region on the spatial light modulator. When the phase shift between the zero-frequency region and the high-frequency region is 0, 2π / 3, or 4π / 3 (e.g. Figure 2 (c) to Figure 2(e)), the interference intensity patterns are I1, I2, and I3, as shown in Figure 3 (c) to 3(e).

[0077] On the CCD surface, the phase-shift interference intensity distribution can be expressed as:

[0078]

[0079] Among them, O0, O d Represent the complex amplitudes of the zero-frequency component and the high-frequency component respectively, and i represents the imaginary part.

[0080] From formula (1), we can get:

[0081]

[0082] If O0 * O d Expressed as in, represents the phase difference between the zero-frequency component and the high-frequency component, then the complex amplitude of the object light wave can be expressed as:

[0083]

[0084] Among them, I0 represents the intensity distribution of the zero-frequency component of the sample. The phase distribution of O0 is ignored here, and the Approximation.

[0085] The imaging performance of the phase contrast microscope device based on partially coherent light illumination of this embodiment is verified by experiments below.

[0086] Experiment 1: Using Figure 1 The quantitative phase contrast microscopy with partially coherent light illumination is shown to image a phase step sample. The phase step (70 μm × 20 μm) is etched on a silicon dioxide slide. Its depth corresponds to a phase of 2.49 rad at a wavelength of 532 nm. Figure 2 (a) to Figure 2 (e) The grayscale image shown in FIG. 2 can obtain the zero-frequency component and high-frequency component of the object light and the different phase-shift interference patterns I1, I2, and I3 on ​​the first CCD camera 21, respectively. Figure 3 (a) to Figure 3 (e) shown.

[0087] See Figure 4 , Figure 4 The quantitative phase contrast microscopy device of the embodiment of the present invention and the existing digital holographic imaging are used to reproduce the phase distribution diagram of the phase steps, wherein: Figure 4 (a) is the phase distribution (rad) of the phase steps reproduced by phase contrast imaging; Figure 4 (b) is the phase distribution (rad) of the phase steps reproduced by digital holographic imaging; Figure 4 (c) Yes Figure 4 (a) and Figure 4 Phase distribution (rad) along the dotted line on the sample in (b).

[0088] The above theoretical method (Formula (1)-(3)) can be used to quantitatively obtain the phase distribution of the sample, such as Figure 4 (a). At the same time, the embodiment of the present invention uses digital holographic microscopy based on optical interference to image the same sample, and the reproduced phase image is as shown in FIG. Figure 4 (b). And Figure 4 (a) and Figure 4 In (b), a dotted line is taken at the same position of the phase step, and the corresponding phase distribution on the two dotted lines is as follows: Figure 4 (c) shows the two curves. By comparing the two curves, we find that two different methods can obtain similar phase distributions. Figure 4 (a) The corresponding phase curve shows that the phase value of the phase step is (2.49±0.15) rad, Figure 4 The corresponding phase curve (b) shows that the phase value of the phase step is (2.49±0.21) rad, which is basically consistent with the actual value of the phase step of 2.49 rad, indicating that the quantitative phase contrast microscopy device of the embodiment of the present invention can perform high-precision quantitative measurement.

[0089] Experiment 2: This experiment compares the quantitative phase contrast microscopy imaging characteristics under coherent illumination and partially coherent illumination. First, the telescope system composed of the sixth lens 19 and the seventh lens 22 is used to image the spectrum of the object light on the spatial light modulator plane onto the second CCD camera 23. When no sample is placed, the spectrum distribution of the HeNe laser illumination and the partially coherent illumination are as follows: Figure 5 (a) and Figure 5 (b). In order to facilitate quantitative comparison, we Figure 5 In (a) and 5(b), two intensity curves passing through the center of the spectrum are taken out respectively, as shown in Figure 5 (c) By performing Gaussian fitting on the two curves, it is found that the full width at half maximum d of the object light spectrum distribution under laser illumination and partially coherent illumination is FWHM Here, the d of the illumination light spectrum distribution is (24.42±0.24)μm and (48.85±0.58)μm respectively. FWHM d corresponds to the zero-frequency component of the object light when the sample is placed FWHM , which is the diameter of the central circular area of ​​the phase mask in phase contrast imaging. Under the partially coherent illumination generated by rotating ground glass and multimode fiber, the d FWHM Satisfaction: d FWHM≤2λ / (ML)f 15 =53.2μm, to prevent the high frequency components of the sample from being mismodulated by the central circular area of ​​the phase mask. For a microscope system with M=10, the imaging field diameter is generally 0.5mm, d FWHM ≤2λ / (ML)f 15 =53.2μm.

[0090] Experiment 3: This experiment compared the coherence noise and illumination uniformity of quantitative phase contrast imaging using partially coherent illumination (PCI) and coherent illumination (CI). Images were captured using two methods: first, CI illumination generated by a HeNe laser coupled into a single-mode fiber (SMF); and second, PCI illumination generated using a twisted ground glass coupled to a multimode fiber. Figure 6 (a) and Figure 6 (b) shows the reconstructed phase image of an optical waveguide sample under CI and PCI illumination. It can be seen from the figure that the PCI image is more uniform than the CI image and has a higher signal-to-noise ratio. In addition, Figure 6 (c) shows Figure 6 (a) and Figure 6 In (b), the phase distribution along the white dashed line (located in the blank area on the sample) shows that the mean square error of the phase curves of PCI and CI is 0.046 and 0.24, respectively. To further quantify the level of coherent noise, the Figure 6 (a) and Figure 6 The phase distribution within the white box (200×180 pixels) in (b) is shown in its phase distribution histogram. Figure 6 (d) and Figure 6 (e) is shown. Figure 6 (d) and Figure 6 The Gaussian fitting of the histogram in (e) shows that the full width at half maximum (FWHM) of the phase distribution under CI illumination is 0.56±0.021, and the full width at half maximum (FWHM) of the phase distribution under PCI illumination is 0.20±0.006, which means that due to the temporal averaging effect of scattered light, the PCI image is more uniform and has lower coherent noise than the CI image.

[0091] In summary, the embodiment of the present invention is based on a quantitative phase contrast microscopy imaging device for partially coherent light illumination. It uses laser as the illumination light source and places a rotating frosted glass sheet before the laser is coupled into the multimode optical fiber, so that the laser forms partially coherent light. The size of the luminous point of the partially coherent light is strictly controlled to the core diameter of the multimode optical fiber through the multimode optical fiber, and the core diameter size is generally between 10 and 100 μm. The optical fiber core is the actual luminous point of the illumination light. When it is imaged onto the spatial light modulator, its diameter d FWHMIt just satisfies the diameter d of the zero-frequency component of the object light calculated by optical diffraction theory. FWHM ≤2λ / (ML)f 15 This effectively prevents the high-frequency components of the sample from being mismodulated by the central circular region of the phase mask. This prevents aliasing of the zero-frequency and high-frequency components of the object light, and also reduces coherent noise.

[0092] In order to reduce the speckle noise of phase contrast imaging and avoid the "halo" phenomenon in phase contrast imaging (ultimately improving the accuracy of phase measurement), our present invention proposes a quantitative phase contrast microscopy technology with partially coherent illumination of small light-emitting surface elements (diameter can be selected in the range of 10-100μm). This technology innovatively uses dynamic scattering and multimode optical fiber to generate partially coherent light illumination with an effective light-emitting point of 10 to 100μm (diameter). The size of this effective light-emitting point is consistent with the size of the zero-frequency component of the object light. The use of a matching circular phase mask will not affect the high-frequency component, and ultimately low-noise, distortion-free quantitative phase contrast imaging can be achieved. In this phase contrast microscopy optical path, the zero-frequency light path and the high-frequency light path pass through exactly the same optical elements, so the phase contrast microscopy device of the present invention has very good immunity to environmental disturbances.

[0093] The quantitative phase contrast microscopy apparatus of the present invention utilizes a spatial light modulator (SLM) loaded with a blazed grating-based mask. This allows for independent measurement of the intensity distributions of the zero-frequency and high-frequency components of the object light and the recording of three phase-shifted interference patterns by introducing different phase values ​​between them. Separately measuring the intensity distributions of the zero-frequency and high-frequency components simplifies the phase reconstruction process, ultimately enabling rapid and accurate quantitative phase distribution of the sample. Furthermore, by digitally determining the size of the zero-frequency component region on the SLM, the contrast of the phase contrast imaging can be modulated.

[0094] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. A quantitative phase contrast microscopy imaging device based on partially coherent light illumination, characterized in that: It includes a partially coherent illumination module, a microscopic imaging system, a spectrum modulation module and an image acquisition module which are sequentially arranged along the optical path, wherein: The partially coherent illumination module is used to generate partially coherent light with an actual luminous point having a size equivalent to the zero-frequency component of the sample as illumination light; The microscopic imaging system is used to obtain a scattered signal of the sample using the partially coherent light and amplify the scattered signal to obtain a light field distribution with sample information; The spectrum modulation module is used to modulate the zero-frequency component and the high-frequency component of the incident light spectrum from the microscopic imaging system; The image acquisition module is used to acquire a hologram generated by the interference of the zero-frequency component and the high-frequency component modulated by the spectrum modulation module; The spectrum modulation module includes a fifth lens (15), a polarizing plate (16), a prism (17) and a spatial light modulator (18), wherein: The polarizer (16) is perpendicular to the direction of the light path and converts light incident on the polarizer (16) into polarized light, and can enable the modulation efficiency of the spatial light modulator (18) to reach a maximum value; The polarized light is incident on the first surface of the prism (17) and then reflected to the spatial light modulator (18) for spectrum modulation, and the modulated spectrum is incident on the second surface of the prism (17) again; The spatial light modulator (18) is located at the rear focal plane of the fifth lens (15) and is used to modulate the zero-frequency component and the high-frequency component of the light with sample information from the microscopic imaging system respectively; by loading a phase mask based on a blazed grating on the spatial light modulator (18), not only can the zero-frequency component and the high-frequency component of the object light be measured separately, but also a phase shift interference pattern between the zero-frequency component and the high-frequency component can be obtained.

2. The quantitative phase contrast microscopy imaging device based on partially coherent light illumination according to claim 1, characterized in that: The partially coherent illumination module comprises a laser (1), and a first plane reflector (2), a first microscope objective lens (3), a frosted glass sheet (4), a first thin lens (5), a second thin lens (6), a multimode optical fiber unit, and a third thin lens (10) arranged in sequence along the optical path of the laser, wherein the frosted glass sheet (4) is arranged perpendicular to the optical axis and can rotate around the optical axis to generate dynamically scattered partially coherent light, and the multimode optical fiber unit is used to collect the dynamically scattered partially coherent light and control the diameter of the partially coherent light; The outlet of the multimode optical fiber unit is located at the focus of the third thin lens (10).

3. The quantitative phase contrast microscopy imaging device based on partially coherent light illumination according to claim 2, characterized in that: 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 the first optical fiber head (7) is located at the focus of the second thin lens (6), and the second optical fiber head (9) is located at the focus of the third thin lens (10).

4. The quantitative phase contrast microscopy imaging device based on partially coherent light illumination according to claim 2, characterized in that: The microscopic imaging system comprises a second microscopic objective lens (12), a fourth lens (13) and a second plane reflector (14) arranged in sequence along an optical path, wherein a sample (11) is placed at the front focal plane of the second microscopic objective lens (12).

5. The quantitative phase contrast microscopy imaging device based on partially coherent light illumination according to claim 4, characterized in that: The spatial light modulator (18) is loaded with a phase mask based on a blazed grating, wherein the phase mask is formed by superimposing a blazed grating and a circular area located at the center of the blazed grating, wherein: The circular area is used to cover and modulate the phase of the spectral components of the object light, and the mask outside the circular area is used to modulate the high-frequency components of the object light. The blazed grating is used to change the propagation direction of the modulated light wave, and can separate the modulated zero-frequency component or high-frequency component of the object light from other spectra. By superimposing different phases in the blazed grating within the circular area, the zero-frequency component of the object light can be phase-delayed to achieve phase-shift contrast imaging.

6. The quantitative phase contrast microscopy imaging device based on partially coherent light illumination according to claim 5, characterized in that: The diameter of the circular area satisfies: ， in, is the wavelength of the illumination light, is the magnification of the telescope system consisting of the second microscope objective (12) and the fourth lens (13), is the focal length of the fifth lens (15), is the diameter of the imaging field of view; The imaging diameter of the output end face of the multimode optical fiber (8) on the spatial light modulator (18) is smaller than the diameter of the circular area.

7. The quantitative phase contrast microscopy imaging device based on partially coherent light illumination according to claim 5, characterized in that: The image acquisition module comprises a sixth lens (19), a beam splitter prism (20), a first CCD camera (21), a seventh lens (22) and a second CCD camera (23), wherein: The sixth lens (19) is perpendicular to the direction of the light path, the reflection surface of the dichroic prism (20) forms a certain angle with the sixth lens (19), the seventh lens (22) is arranged in the light reflection direction of the dichroic prism (20), and the second CCD camera (23) is located at the back focal plane of the seventh lens (22); The first CCD camera (21) is located in the light transmission direction of the beam splitter prism (20).

8. The quantitative phase contrast microscopy imaging device based on partially coherent light illumination according to claim 7, characterized in that: A grayscale grating image is loaded on the central circular area of ​​the spatial light modulator (18) to obtain the zero-frequency component intensity distribution of the object light on the first CCD camera (21) ; A grayscale grating image is loaded on the blazed grating area around the spatial light modulator (18) to obtain the high-frequency component intensity distribution of the object light on the first CCD camera (21) ; The central circular area and the surrounding blazed grating area of ​​the spatial light modulator (18) are loaded with 0, 、 The grayscale grating pattern of the phase shift amount is formed to obtain the interference intensity image of the object light on the first CCD camera (21) 、 、 .

9. The quantitative phase contrast microscopy imaging device based on partially coherent light illumination according to claim 8, characterized in that: Also includes a data processing module for determining the intensity distribution of the zero-frequency component of the object light And the interference intensity image of the object light 、 、 Get the complex amplitude of the object light wave, Specifically, the interference intensity image of the object light is obtained 、 、 The expression: in, 、 represent the complex amplitudes of the zero-frequency component and the high-frequency component, respectively. i represents the imaginary part; Further we get: Will Expressed as ,in, represents the phase difference between the zero-frequency component and the high-frequency component, then the complex amplitude of the object light wave is expressed as: in, Represents the intensity distribution of the zero-frequency component of the sample.

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