WO2023221741A1 - 一种基于非干涉合成孔径的光强传输衍射层析显微成像方法 - Google Patents

一种基于非干涉合成孔径的光强传输衍射层析显微成像方法 Download PDF

Info

Publication number
WO2023221741A1
WO2023221741A1 PCT/CN2023/090075 CN2023090075W WO2023221741A1 WO 2023221741 A1 WO2023221741 A1 WO 2023221741A1 CN 2023090075 W CN2023090075 W CN 2023090075W WO 2023221741 A1 WO2023221741 A1 WO 2023221741A1
Authority
WO
WIPO (PCT)
Prior art keywords
dimensional
spectrum
imaging
synthetic aperture
sample
Prior art date
Application number
PCT/CN2023/090075
Other languages
English (en)
French (fr)
Inventor
左超
陈钱
孙佳嵩
冯世杰
张玉珍
李加基
周顺
Original Assignee
南京理工大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京理工大学 filed Critical 南京理工大学
Publication of WO2023221741A1 publication Critical patent/WO2023221741A1/zh

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation

Definitions

  • the invention belongs to optical microscopic measurement and three-dimensional refractive index imaging technology, especially a light intensity transmission diffraction tomography microscopic imaging method based on non-interference synthetic aperture.
  • fluorescence microscopy imaging modalities such as widefield, confocal, total internal reflection fluorescence, two/multiphoton and light-sheet fluorescence microscopy techniques, which are used to detect very weak signals and reveal Powerful tool for 3D structural and functional characterization of fixed or living cells with high specificity.
  • fluorescent markers attached to specific molecular structures are excited by short-wavelength lasers and radiate long-wavelength fluorescence, allowing imaging of otherwise transparent biological samples.
  • super-resolution fluorescence microscopy technology has broken through the diffraction limit and increased the imaging resolution to tens of nanometers, providing technical means for research at the subcellular scale.
  • phase contrast microscopy uses refractive index as an intrinsic optical imaging Contrast, label-free imaging of biological samples without the use of exogenous labeling agents.
  • the measured data is only the light absorption or optical path difference accumulation along the axial direction of the object to be measured.
  • the refractive index and thickness information reflecting the sample information are coupled with each other, and three-dimensional information cannot be obtained.
  • label-free three-dimensional imaging of biological samples has become a popular research direction.
  • optical holography made it possible to measure small phase differences caused by samples, promoting phase
  • various optical diffraction tomography methods have been developed to infer the three-dimensional refractive index distribution of biological samples through object rotation or illumination scanning.
  • optical diffraction chromatography makes three-dimensional label-free microscopy possible and has been successfully used to study various types of biological samples such as blood cells, neuronal cells, cancer cells, and bacteria.
  • interference-based optical diffraction tomography usually uses temporally coherent illumination sources, which results in speckle noise in the imaging results, hindering the formation of high-quality images.
  • most of them require the use of interference devices with complex beam scanning devices, which hinders their wide application in biological and medical fields.
  • the object of the present invention is to provide a light intensity transmission diffraction tomography microscopic imaging method based on non-interference synthetic aperture.
  • the technical solution to achieve the purpose of the present invention is: a light intensity transmission diffraction tomography microscopic imaging method based on non-interference synthetic aperture, the steps are as follows:
  • Step 1 Collect axial defocus intensity stacks at different illumination angles
  • Step 2 Calculate the three-dimensional logarithmic intensity spectrum under different incident illumination, and perform three-dimensional half-space Fourier filtering on each logarithmic intensity spectrum to obtain a three-dimensional scattering field containing the real and imaginary parts of the complex phase function under different incident illumination. , synthesize all single-sideband three-dimensional scattering fields in Fourier space to achieve non-interference synthetic holes diameter to obtain a preliminary estimate of the three-dimensional scattering potential spectrum of the sample;
  • Step 3 Perform three-dimensional deconvolution based on LED discrete sampling, illumination partial coherence and correction factors on the initially estimated spectrum
  • Step 4 Use a hybrid iterative constraint algorithm that combines non-negative constraints and total variation regularization to calculate and fill in the missing cone information in the synthesized scattering potential spectrum;
  • Step 5 Perform a three-dimensional inverse Fourier transform on the filled three-dimensional scattering potential spectrum to restore the three-dimensional refractive index distribution of the sample to achieve non-invasive three-dimensional imaging of label-free biological samples.
  • a light intensity transmission diffraction tomography microscopy imaging platform based on non-interference synthetic apertures is used to collect axial defocus intensity stacks at different illumination angles.
  • the light intensity transmission diffraction tomography microscopy imaging platform includes a programmable LED. Array, electric displacement stage scanning device, sample to be tested, microscope objective, imaging tube lens and camera.
  • the center of the programmable LED array coincides with the optical axis of the microscope objective and is placed at a set distance from the sample.
  • the back focal plane coincides with the front focal plane of the tube lens, and the imaging plane of the camera is placed at the back focal surface of the imaging tube lens.
  • the sample is placed on the electric displacement stage, the LED units are lit one by one, and the quasi-monochromatic plane wave is irradiated.
  • the measured sample passes through the objective lens and is converged by the imaging tube lens before falling on the imaging plane of the camera.
  • the camera is used to record the three-dimensional light intensity stack.
  • step 2 the logarithm of the three-dimensional intensity stack under different incident illumination is taken, and a three-dimensional Fourier transform is performed to obtain a three-dimensional logarithmic intensity spectrum.
  • ⁇ (r) and a(r) correspond to the phase and absorption of the scattering potential O(r)
  • g(r) and g′(r) are the point spread function of the tomography system and the corresponding incident light, respectively.
  • U in (r) modulated point spread function, g′ * (r) is the conjugate form of g′(r);
  • the absorption and phase transfer functions of the diffraction tomography system are respectively expressed as:
  • u (u T ,u z ) is the spatial frequency coordinate corresponding to r
  • um n m / ⁇
  • n m the refractive index of the medium surrounding the sample
  • ⁇ is the illumination in free space Wavelength
  • P * (u) is the conjugate form of P (u)
  • P (u + u in ) and P * (uu in ) are the translations of P (u) and P * (u) by the incident light spatial frequency u in respectively.
  • three-dimensional half-space Fourier filtering or three-dimensional Hilbert transform is performed on each logarithmic intensity spectrum to obtain a three-dimensional scattering field containing the real part and the imaginary part of the complex phase function under different incident illumination, in Fourier space
  • the specific process of synthesizing all single sideband three-dimensional scattering fields, achieving non-interference synthetic aperture, and obtaining a preliminary estimate of the three-dimensional scattering potential spectrum of the sample is:
  • the scattering potential spectrum after translational modulation by the spatial frequency u in of the incident light is the generalized coherent transfer function of the system, and its limited support domain is called the Ewald spherical shell.
  • the deconvolution process in step 3 is expressed as:
  • H syn is the synthetic Three-dimensional incoherent transfer function of the system after aperture processing, is the conjugate form of H syn , and ⁇ is the regularization parameter.
  • the three-dimensional incoherent transfer function of the system after synthetic aperture processing is specifically:
  • j is the imaginary unit
  • ⁇ is the illumination wavelength in free space
  • P(u T ) represents the objective lens pupil function, that is, the two-dimensional coherent transfer function. Ideally, it is a circular function with a radius of NA obj / ⁇ , determined by the objective lens.
  • NA obj is determined.
  • u T (u x ,u y )
  • S is the spatial frequency intensity distribution of the illumination light source. function.
  • the present invention has significant advantages:
  • the first-order scattering fields under different illumination angles are synthesized in the three-dimensional spectral space, which expands the spectral information accessible to the sample and greatly improves the imaging resolution and optical sectioning capabilities.
  • the system's full-width lateral resolution is 330 nm, and the axial resolution is 1.58 ⁇ m;
  • the system's full-width lateral resolution reaches 206 nm.
  • the axial resolution reaches 0.52 ⁇ m.
  • the data acquisition time can be shortened and rapid long-term imaging of dynamic samples can be achieved.
  • Figure 1 is a flow chart of the light intensity transmission diffraction tomography microscopic imaging method based on non-interference synthetic aperture.
  • Figure 2 is a schematic diagram of the light intensity transmission diffraction tomography microscopic imaging illumination system based on non-interference synthetic aperture as well as a synchronization block diagram of the hardware platform and electromechanical system.
  • Figure 3 is a synchronized time series of data acquisition cycles for the light intensity transmission diffraction tomography microscopic imaging method based on non-interference synthetic aperture.
  • Figure 4 is an analytical analysis of the two-dimensional spectrum and the three-dimensional spectrum under different lighting numerical apertures.
  • Figure 5 is a data processing flow chart for three-dimensional refractive index reconstruction using the light intensity transmission diffraction tomography microscopic imaging method based on non-interference synthetic aperture, taking unstained MCF-7 cells as an example.
  • the concept of the present invention is: a light intensity transmission diffraction tomography microscopic imaging method based on non-interference synthetic aperture, by collecting axial defocus intensity stacks under different illumination angles, and performing a half-space Fu on the logarithmic light intensity spectrum.
  • Liye filtering or equivalent three-dimensional Hilbert transform combined with non-interference synthetic aperture, enables diffraction tomography based on non-interference measurement without matching illumination conditions. Due to the inherent synthetic aperture advantage, the imaging resolution reaches the incoherent imaging diffraction limit, and high-resolution imaging results are obtained.
  • the imaging optical path is simple and stable, the imaging results are not affected by speckle and parasitic interference, and it is highly compatible with traditional brightfield microscope structures.
  • Step 1 Collect axial defocus intensity stacks at different illumination angles
  • This step designs a reasonable synchronization mode, which effectively coordinates the timing control between LED lighting mode switching, electric displacement stage axial scanning and camera reading, and achieves fine and stable acquisition of axial defocus intensity image stacks under different lighting angles. .
  • the present invention is a light intensity transmission diffraction tomography microscopic imaging system based on non-interference synthetic aperture.
  • the actual hardware platform of the system includes a programmable LED array (such as a programmable multi-ring LED array), an electric displacement stage scanning Device, sample to be tested, microscope objective lens, imaging tube lens and camera.
  • a programmable LED array such as a programmable multi-ring LED array
  • an electric displacement stage scanning Device sample to be tested
  • microscope objective lens imaging tube lens
  • imaging tube lens imaging tube lens
  • camera an example of a schematic diagram of the imaging platform lighting system and a synchronization block diagram of the hardware platform and electromechanical system is given.
  • the programmable multi-ring LED array includes a total of 128 LED units, which are distributed on five concentric rings with different radii, and are arranged at equal intervals on each ring.
  • Each LED unit is a red, green, and blue LED unit with typical wavelengths of red light 629nm, green light 520nm, and blue light 483nm.
  • the multi-ring LED array does not need to be processed separately and can generally be purchased directly on the market.
  • Table 1 gives a Product parameters of available LED arrays.
  • the center of the multi-ring LED array coincides with the optical axis of the microscope objective and is placed 25mm away from the sample to provide quasi-monochromatic plane wave illumination with a variable illumination angle of about 72°, corresponding to the maximum illumination numerical aperture. is 0.95.
  • the back focal plane of the microscope objective coincides with the front focal plane of the tube lens, and the imaging plane of the camera is placed at the back focal plane of the imaging tube lens.
  • the quasi-monochromatic plane wave irradiates the sample to be measured, passes through the objective lens, is condensed by the imaging tube lens, and then falls on the imaging plane of the camera.
  • Directional scanning uses cameras to record three-dimensional light intensity stacks.
  • Each LED unit in the LED array can be lit individually and turned on sequentially under the control of a hardware control circuit (such as an ARM control board).
  • the data acquisition computer software communicates with the camera and electric displacement stage through programming interfaces and programs.
  • the camera and LED array are synchronized through the same controller using two coaxial cables to provide triggering and monitoring of exposure status.
  • the hardware control circuit provides a series of The trigger signal is used to control camera triggering.
  • software (such as ⁇ -Manager) is used to control a high-precision electric displacement stage to scan different focal planes.
  • the software transmits drive signals and steps to complete markings synchronously with the hardware control circuit through USB slave mode.
  • this method uses a fine time sequence to synchronize the movement of the electric stage and the exposure of the camera. It uses the external triggering method of the camera, combined with the hardware control circuit, to synchronize the switching of the LED lights during the row exposure. control.
  • the periodic synchronization time sequence between LED illumination mode switching, motorized stage axial scanning and camera reading is shown in Figure 4. Due to the synchronization between each intensity stack exposure sequence and the LED angle illumination, this mode is equivalent to using a global shutter mode. In addition, by reducing the equivalent exposure time (for example, controlling the exposure time to around 50ms), vibration noise and time-varying motion artifacts are minimized. In order to avoid changes in the actual exposure time and instability in the initial state of the focused scanning phase, this method requires a delay before acquiring a new set of intensity stacks.
  • this method can reduce the resistance of the current output limiting resistor to provide sufficient total photon flux under the same exposure time.
  • the data acquisition time is shortened to reach the imaging speed limit of the system to meet the imaging needs of dynamic samples. For example, an intensity stack containing a total of 12 different illumination angles containing at least 15 different axial displacement data frames can be captured within 20 seconds.
  • Step 2 Take the logarithm of the three-dimensional intensity stack under different incident illumination and perform a three-dimensional Fourier transform to obtain a three-dimensional logarithmic intensity spectrum; then perform three-dimensional half-space Fourier filtering on each logarithmic three-dimensional intensity spectrum Or equivalently, perform a three-dimensional Hilbert transform to obtain a three-dimensional scattering field containing the real and imaginary parts of the complex phase function under different incident illumination, and then synthesize all single-sideband three-dimensional scattering fields in Fourier space to achieve non-interference synthesis. aperture to obtain a preliminary estimate of the object's three-dimensional scattering potential spectrum.
  • the specific implementation process is: for volume imaging of three-dimensional thick objects, the scattering potential function O(r) is commonly used to characterize the three-dimensional structure of the sample.
  • ⁇ (r) and a(r) correspond to the phase and absorption of the scattering potential O(r)
  • g(r) and g′(r) are the point spread function of the tomography system and the corresponding incident light, respectively.
  • U in (r) modulated point spread function, g′ * (r) is the conjugate form of g′(r).
  • H a (u) and H ⁇ (u) are The absorption and phase transfer functions of the diffraction tomography system are respectively expressed as:
  • each double-sideband three-dimensional spectrum is subjected to three-dimensional half-space Fourier filtering or equivalently three-dimensional Hilbert transform, and the complex phase function under different incident illumination can be obtained.
  • Step 3 in order to compensate for the effects of LED element discretization and illumination partial coherence (time and space), a three-dimensional deconvolution based on LED discrete sampling, illumination partial coherence and correction factors is further performed on the preliminary synthesized spectrum in the method.
  • the specific implementation process is: in order to compensate for the effects of LED element discretization and illumination partial coherence (time and space), three-dimensional deconvolution is further performed on the preliminary synthesized spectrum. This deconvolution is based on discrete sampling of LEDs and illumination partial coherence.
  • the deconvolution process of the transfer function taking into account the properties and correction factors is expressed as:
  • H syn is the three-dimensional incoherent transfer function of the system after synthetic aperture processing, is the conjugated form of H syn , and ⁇ is Regularization parameters.
  • the three-dimensional incoherent transfer function H syn of the system can be expressed as:
  • j is the imaginary unit
  • ⁇ is the illumination wavelength in free space
  • P(u T ) represents the objective pupil function, that is, the two-dimensional coherent transfer function.
  • NA obj is determined.
  • u T (u x , u y ) is the two-dimensional spatial frequency coordinate.
  • S is the spatial frequency intensity distribution function of the lighting source.
  • the purpose of adding regularization parameters is to prevent excessive amplification of noise during the deconvolution process.
  • deconvolution performance strongly depends on the choice of regularization parameters, while the signal-to-noise ratio of the intensity stack also affects the quality of the final tomography result.
  • the regularization parameters of the dry mirror and oil mirror can be set to around 0.1 and 0.25 respectively.
  • Step 4 Use a hybrid iterative constraint algorithm that combines non-negative constraints and total variation regularization to calculate and fill in the missing cone information in the synthesized scattering potential spectrum.
  • the specific implementation process is: based on the prior knowledge of the sample, in the air domain, it is considered that the refractive index of the sample is always higher than the refractive index of the medium, and the gradient value is used as the optimization objective function, and in the frequency domain, the experimentally measured spectrum is determined
  • the data is real and valid.
  • Step 5 Perform a three-dimensional inverse Fourier transform on the final synthesized three-dimensional scattering potential spectrum to restore the three-dimensional refractive index distribution of the sample to be tested.
  • the specific implementation process is as follows: perform inverse Fourier transform on the three-dimensional scattering potential spectrum obtained in step 5 to obtain the three-dimensional scattering potential O(r).
  • the real part represents the refractive index of the sample
  • the imaginary part represents the sample. Absorption.
  • the present invention uses both a programmable LED array and an electric displacement stage.
  • the LED array serves as the lighting source to ensure that the lighting mode is programmable and controllable, and is used to provide the required quasi-monochromatic plane wave lighting with variable lighting angles.
  • the electric displacement stage is controlled by software and cooperates with the timing signal between the LED array and camera exposure to provide nanometer-level axial displacement of the sample, realizing four-dimensional data collection of the three-dimensional light intensity image stack of the sample under different illumination angles.
  • the first-order scattered fields under different illumination angles are spliced and synthesized in a three-dimensional spectrum.
  • the imaging resolution reaches the incoherent diffraction limit, and the imaging results are not affected by speckle and parasitic interference.

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

一种基于非干涉合成孔径的光强传输衍射层析显微成像方法，通过采集不同照角度下的轴向离焦强度堆栈，在光强频谱上执行半空间傅里叶滤波或等效的三维希尔伯特变换，结合非干涉合成孔径，实现了基于非干涉测量下无需满足匹配照明条件的衍射层析成像。由于固有的合成孔径优势，使得成像分辨率达到非相干成像衍射极限，获得了高分辨率成像结果。采用非干涉测量，成像光路简单，光学路径稳定，成像结果不受散斑和寄生干涉影响，并且可高度兼容传统明场显微镜结构。

Description

一种基于非干涉合成孔径的光强传输衍射层析显微成像方法 技术领域
本发明属于光学显微测量、三维折射率成像技术，特别是一种基于非干涉合成孔径的光强传输衍射层析显微成像方法。
背景技术
在生物医学显微成像领域，大部分活细胞和未染色的生物标本都是无色透明的，这是因为细胞内各部分细微结构的折射率和厚度不同，当光波通过时，波长和振幅并不发生变化，仅相位发生变化，但这种相位差人眼无法观察。这就需要通过一些化学或者生物手段来对细胞进行染色或标记，从而使其在显微镜下可见。在过去的几十年里，开发了多种荧光显微成像方式，如宽场、共聚焦、全内反射荧光、双/多光子和光片荧光显微镜技术，它们被作为探测非常微弱的信号和揭示固定或活细胞的三维结构和功能特性的强大工具，具有很高的特异性。在这些技术中，附着在特定分子结构上的荧光标记物被短波长激光激发后辐射出长波长荧光，从而可对原本透明的生物样本进行成像。进入21世纪以来，超分辨荧光显微技术突破了衍射极限,将成像分辨率提升至几十纳米，为亚细胞尺度的研究提供了技术手段。目前的超分辨荧光显微成像方法有受激发射损耗显微成像(STED)、结构光照明显微术(SIM)、随机光学重建显微术(STORM)以及光激活定位显微术(PLAM)等。然而，这些技术不适合成像非荧光样品，或可视化不能被荧光分子标记的细胞成分，从而限制了荧光显微技术的应用范围。此外，外源性荧光剂带来的光毒性可能会对细胞活性等细胞功能产生不可逆的负面影响，而相关的光漂白性会在一段较长的时间内阻止活细胞长时间成像。
近年来，为了简化样本制备过程、消除荧光分子对待测样品的干扰并满足临床的成像需求，无标记光学成像成为了生物医学显微成像研究的热点，相衬显微镜利用折射率作为本征光学成像对比度，在不使用外源性标记剂的情况下对生物样品进行无标记成像。其中二维无标记成像，测得的数据只是待测物体沿轴向的光吸收或光程差积累，反映样品信息的折射率与厚度信息相互耦合，无法得到三维信息。为了获得更准确的形态学信息，如体积、形状、干质量等，生物样本的无标记三维成像成为目前研究的一大热门方向。
光学全息术的引入使得测量由样品引起的微小相位差成为可能，促进了相位 成像技术从定性观察到定量测量的发展。将光学全息术与计算机断层扫描相结合，通过物体旋转或照明扫描，目前已经开发出了各种光学衍射层析成像方法用以推断生物样品的三维折射率分布。特别是光学衍射层析使三维无标记显微镜成为可能，并已成功应用于研究血细胞、神经元细胞、癌细胞、细菌等各种类型的生物样品。然而，基于干涉的光学衍射层析通常使用时间相干照明光源，使得成像结果中存在散斑噪声，阻碍了高质量图像的形成。此外，它们大多数都需要采用具有复杂光束扫描装置的干涉装置，这妨碍了它们在生物和医学领域的广泛应用。
为了弥补基于干涉测量的光学衍射层析成像方式的缺点和不足，推动三维折射率成像在生物医药领域中的应用，各种基于非干涉测量的层析成像技术在近几年逐渐发展起来。由于采用基于非干涉的测量方式，相机上只有散射场的光强数据被记录，而相位信息完全丢失，因此无论对于二维定量相位成像还是三维衍射层析成像，所有基于聚焦探测的非干涉测量成像方式都需要满足匹配照明条件(照明数值孔径等于物镜数值孔径)，才能实现相位或折射率信息的正确恢复。然而，在实际应用中很难严格实现匹配照明条件，特别是对于高数值孔径显微系统，若采用油浸物镜，则必须借助聚光镜才有可能实现照明条件的匹配。在不满足匹配照明条件的情况下，由于捕获的强度频谱中低频频谱存在重叠，导致无法完整恢复相位分量，也就是说，不能正确恢复样品的三维折射率。所以，如何规避匹配照明条件，即在任意照明下都能实现基于非干涉测量的衍射层析成像，精确重建待测样品的三维折射率分布，并且能达到非相干衍射极限成像分辨率是一大技术难题。
发明内容
本发明的目的在于提供一种基于非干涉合成孔径的光强传输衍射层析显微成像方法。
实现本发明目的的技术解决方案为：一种基于非干涉合成孔径的光强传输衍射层析显微成像方法，步骤如下：
步骤1，采集不同照角度下的轴向离焦强度堆栈；
步骤2：计算不同入射光照下的三维对数强度谱，并对每个对数强度谱进行三维半空间傅里叶滤波，获得不同入射光照下包含复相位函数实部和虚部的三维散射场，在傅里叶空间合成所有单边带三维散射场，实现非干涉合成孔 径，得到样品三维散射势频谱的初步估计；
步骤3：对初步估计的频谱进行基于LED离散采样、照明部分相干和修正因子的三维反卷积；
步骤4：采用非负约束和全变分正则化相结合的混合迭代约束算法对合成的散射势频谱中缺失锥信息进行计算填充；
步骤5：对填充后的的三维散射势频谱进行三维傅里叶逆变换，恢复样品的三维折射率分布，实现无标记生物样品实现非侵入式三维成像。
优选地，利用基于非干涉合成孔径的光强传输衍射层析显微成像平台采集不同照角度下的轴向离焦强度堆栈，所述光强传输衍射层析显微成像平台包括包括可编程LED阵列、电动位移台扫描装置，待测样品、显微物镜、成像筒镜和相机，可编程LED阵列的圆心和显微物镜光轴重合，被放置在距离样品设置距离的位置，显微物镜的后焦面与筒镜的前焦面重合，相机的成像平面放置在成像筒镜的后焦面位置，成像时样品被放置在电动位移台上，逐个点亮LED单元，准单色平面波照射待测样品，通过物镜，经过成像筒镜汇聚后落在相机的成像平面上，通过控制电动位移台的轴向扫描，利用相机来记录三维光强堆栈。
优选地，步骤2中对不同入射光照下的三维强度堆栈取对数，并进行三维傅里叶变换，得到三维对数强度谱。
优选地，采用散射势函数O(r)来表征样品的三维结构，将散射势函数O(r)展开成实部和虚部的形式，即O(r)＝a(r)+jφ(r)，其中φ(r)和a(r)对应于散射势O(r)的相位成分和吸收成分；
对不同入射光照下的三维强度堆栈I(r)取对数，表示为：
式中，φ(r)和a(r)对应于散射势O(r)的相位和吸收，g(r)和g′(r)分别为层析成像系统的点扩散函数和对应的受入射光Uin(r)调制后的点扩散函数，g′*(r)是g′(r)的共轭形式；
通过计算上式的傅里叶变换，得到对数强度谱函数：
式中的和分别对应于三维强度堆栈I(r)、散射势O(r)的吸收 成分a(r)和相位成分φ(r)的三维傅里叶变换，Ha(u)和Hφ(u)为衍射层析成像系统的吸收和相位传递函数。
优选地，衍射层析成像系统的吸收和相位传递函数分别表示为：
式中，为系统的广义相干传递函数，u＝(uT,uz)是对应于r的空间频率坐标，um＝nm/λ，nm为样品周围介质折射率，λ为自由空间中的照明波长，P*(u)是P(u)的共轭形式，P(u+uin)和P*(u-uin)分别为P(u)和P*(u)受入射光空间频率uin平移调制后的相干传递函数表达式。
优选地，对每个对数强度谱进行三维半空间傅里叶滤波或三维希尔伯特变换，获得不同入射光照下包含复相位函数实部和虚部的三维散射场，在傅里叶空间合成所有单边带三维散射场，实现非干涉合成孔径，得到样品三维散射势频谱的初步估计的具体过程为：
根据频谱中两个反对称广义孔径的位置，对每个双边带三维光谱进行三维半空间傅里叶滤波或三维希尔伯特变换，得到不同入射光照下包含复相位函数实部和虚部的三维散射场Us1(r)，根据傅里叶衍射定理
在傅里叶空间合成所有单边带三维散射场，实现非干涉合成孔径，得到物体三维散射势频谱的初步估计，式中u＝(uT,uz)是对应于r的空间频率坐标，j是虚数单位，和分别对应于O和Us1的傅里叶变换，是受入射光空间频率uin平移调制后的散射势频谱，为系统的广义相干传递函数，其有限支持域被称为Ewald球壳。
优选地，步骤3中反卷积过程表示为：
其中和分别样品散射势的最终反卷积频谱和初步合成频谱，Hsyn为合成 孔径处理后系统的三维非相干传递函数，是Hsyn的共轭形式，ε为正则化参数。
优选地，合成孔径处理后系统的三维非相干传递函数具体为：
其中j是虚数单位，λ为自由空间中的照明波长，P(uT)代表物镜光瞳函数，即二维相干传递函数，理想情况下是一个半径为NAobj/λ的圆函数，由物镜数值孔径NAobj决定，u＝(uT,uz)是对应于r的空间频率坐标，uT＝(ux,uy)是二维空间频率坐标，S是照明光源的空间频率强度分布函数。
本发明与现有技术相比，其显著优点：
(1)基于非干涉测量的衍射层析成像技术无需引入复杂且不稳定的干涉光路和装置，使得实验装置简单，易于和传统明场显微镜相结合。
(2)采用准单色LED照明光源，避免了高时间相干性激光光源带来的散斑噪声和寄生干涉，提高了成像质量。
(3)将光强传输从二维平面传输拓展到三维体传输，通过在对数强度谱上执行三维半空间滤波或等效的希尔伯特变换，可以在仅强度测量下实现散射场复相位(振幅和相位)的完整信息恢复。最终实现无需匹配照明条件的仅光强数据测量的衍射层析成像，正确恢复待测样品的三维折射率。
(4)通过合成孔径，将不同照明角度下的一阶散射场在三维频谱空间中合成，拓展了样品可访问的频谱信息，极大程度地提高的成像分辨率和光学切片能力。例如在40倍0.95数值孔径的干镜下，系统全宽横向分辨率为330nm，轴向分辨率为1.58μm；在100倍1.4数值孔径的油浸物镜下，系统全宽横向分辨率达到206nm，轴向分辨率达到0.52μm。
(5)通过照明角度和轴向离焦切片数的降采样，可以缩短数据采集时间，实现对动态样品的快速长时间成像。
下面结合附图对本发明作进一步详细描述。
附图说明
图1是基于非干涉合成孔径的光强传输衍射层析显微成像方法的流程图。
图2是基于非干涉合成孔径的光强传输衍射层析显微成像照明系统示意图以及硬件平台和机电系统同步框图。
图3是基于非干涉合成孔径的光强传输衍射层析显微成像方法的数据采集周期同步时间序列。
图4是不同照明数值孔径下二维频谱和三维频谱的解析性分析。
图5为以未染色MCF-7细胞为例，应用基于非干涉合成孔径的光强传输衍射层析显微成像方法进行三维折射率重建的数据处理流程图。
具体实施方式
本发明的构思为：一种基于非干涉合成孔径的光强传输衍射层析显微成像方法，通过采集不同照角度下的轴向离焦强度堆栈，在对数光强频谱上执行半空间傅里叶滤波或等效的三维希尔伯特变换，结合非干涉合成孔径，从而实现了基于非干涉测量下无需满足匹配照明条件的衍射层析成像。由于固有的合成孔径优势，使得成像分辨率达到非相干成像衍射极限，获得了高分辨率成像结果。采用非干涉测量，成像光路简单，光学路径稳定，成像结果不受散斑和寄生干涉影响，并且可高度兼容传统明场显微镜结构。
如图1所示，一种基于非干涉合成孔径的光强传输衍射层析显微成像方法，具体步骤为：
步骤1，采集不同照角度下的轴向离焦强度堆栈；
本步骤设计了合理的同步模式，有效协调了LED照明模式切换、电动位移台轴向扫描和相机读取之间的时序控制，实现了不同照明角度下轴向离焦强度图像堆栈的精细稳定采集。
具体实施过程为：本发明是基于非干涉合成孔径的光强传输衍射层析显微成像系统，该系统的实际硬件平台包括可编程LED阵列(例如可编程多环LED阵列)、电动位移台扫描装置，待测样品、显微物镜、成像筒镜和相机。如图2所示，给出了成像平台照明系统示意图以及硬件平台和机电系统同步框图的示例。在示例中可编程多环LED阵列共包括128个LED单元，分别分布在五个半径不同的同心圆环上，在每个圆环上等间距排列。其中每个LED单元均为红、绿、蓝三色LED单元，其典型波长为红光629、绿光520和蓝光483nm。该多环LED阵列并不需要进行单独加工，一般在市场上可直接购置，表1给出了一个市面上 可购置的LED阵列的产品参数。
表1可编程多环LED的物理参数
其中多环LED阵列的圆心和显微物镜光轴重合，被放置在距离样品25mm的位置，提供最大照明角度约为72°的可变照明角度的准单色平面波照明，对应最大的照明数值孔径为0.95。显微物镜的后焦面与筒镜的前焦面重合，相机的成像平面放置在成像筒镜的后焦面位置。成像时样品被放置在电动位移台上，逐个点亮LED单元，准单色平面波照射待测样品，通过物镜，经过成像筒镜汇聚后落在相机的成像平面上，通过控制电动位移台的轴向扫描，利用相机来记录三维光强堆栈。
LED阵列中每个LED单元均可被单独点亮，由硬件控制电路(例如ARM控制板)控制顺序打开。数据采集计算机软件通过编程接口和程序与相机、电动位移台器进行通信，相机与LED阵列通过同一控制器，利用两根同轴电缆进行同步，提供触发和监测曝光状态，硬件控制电路提供一系列触发信号用于控制相机触发。在一定的光照角度下，利用软件(例如μ-Manager)控制高精度电动位移台对不同焦平面进行扫描，该软件通过USB从机模式与硬件控制电路同步传输驱动信号和步进完成标记。为了最小化采集时间，该方法使用了一个精细的时间序列来同步电动位移台的运动和相机的曝光，利用相机的外置触发方式，结合硬件控制电路，对行曝光时LED灯的切换进行同步控制。LED照明模式切换、电动位移台轴向扫描和相机读取之间的周期同步时间序列如图4所示。由于每个强度堆栈曝光序列与LED角度照明之间的同步，该模式相当于使用全局快门模式。此外，通过减少等效曝光时间(例如控制曝光时间在50ms左右)，最小化了振动噪声和随时间变化的运动伪影吗。为了避免实际曝光时间的变化和聚焦扫描阶段初始状态的不稳定，该方法在获取新一组强度堆栈前需延迟一段时间。
对于动态样品的长时间成像，该方法可减小电流输出限制电阻的阻值，以相同的曝光时间下提供足够的总光子通量。通过降采样照明角度和z轴离焦切片数，缩短数据采集时间，以达到系统的成像速度极限来满足对动态样品的成像需求。例如可在20秒内捕获包含至少15个不同轴向位移数据帧的共12个不同照明角度下的强度堆栈。
步骤2，对不同入射光照下的三维强度堆栈取对数，并进行三维傅里叶变换，即可得到三维对数强度谱；然后对每个对数三维强度谱进行三维半空间傅里叶滤波或等价于执行三维希尔伯特变换，得到不同入射光照下包含复相位函数实部和虚部的三维散射场，接着在傅里叶空间合成所有单边带三维散射场，实现非干涉合成孔径，得到物体三维散射势频谱的初步估计。
具体实施过程为：对于三维厚物体的体成像，常用散射势函数O(r)来表征样品的三维结构，O(r)可展开成实部和虚部的形式，即O(r)＝a(r)+jφ(r)，其中φ(r)和a(r)对应于散射势O(r)的相位成分和吸收成分。
对不同入射光照下的三维强度堆栈I(r)取对数，表示为：
式中，φ(r)和a(r)对应于散射势O(r)的相位和吸收，g(r)和g′(r)分别为层析成像系统的点扩散函数和对应的受入射光Uin(r)调制后的点扩散函数，g′*(r)是g′(r)的共轭形式。
通过计算上式的傅里叶变换，得到对数强度谱函数：
式中的和分别对应于三维强度堆栈I(r)，散射势O(r)的吸收成分a(r)和相位成分φ(r)的三维傅里叶变换，Ha(u)和Hφ(u)为衍射层析成像系统的吸收和相位传递函数，分别表示为：
式中为系统的广义相干传递函数，u＝(uT,uz)是对应于r的空间频率坐标，um＝nm/λ，nm为样品周围介质折射率，λ为自由空 间中的照明波长，P*(u)是P(u)的共轭形式。P(u+uin)和P*(u-uin)分别为P(u)和P*(u)受入射光空间频率uin平移调制后的相干传递函数表达式。
因此，在三维对数强度谱的ux-uz截面上可以清晰地观察到两个反对称广义孔径，这两个孔径根据入射光的角度发生位移，并且在三维空间中镜像对称地移动，它们永远不会相互抵消，如图四所示。根据频谱中两个反对称广义孔径的位置，对每个双边带三维光谱进行三维半空间傅里叶滤波或等效地执行三维希尔伯特变换，就可以得到不同入射光照下包含复相位函数实部和虚部的三维散射场Us1(r)的单边带频谱。
根据傅里叶衍射定理
在傅里叶空间合成所有单边带三维散射场，实现非干涉合成孔径，就可以得到物体三维散射势频谱的初步估计，式中u＝(uT,uz)是对应于r的空间频率坐标，j是虚数单位，和分别对应于O和Us1的傅里叶变换，是受入射光空间频率uin平移调制后的散射势频谱，为系统的广义相干传递函数，其有限支持域被称为Ewald球壳。如图5所示，给出了以未染色MCF-7细胞为例的重建数据处理流程图。
步骤3，为了补偿LED元件离散化和照明部分相干性(时间和空间)的影响，在方法中进一步对初步合成的频谱执行基于LED离散采样、照明部分相干和修正因子的三维反卷积。
具体实施过程为：为了补偿LED元件离散化和照明部分相干(时间和空间)的影响，进一步对初步合成的光谱进行了三维反卷积，该反卷积是基于将LED离散采样、照明部分相干性、修正因子考虑在内的传递函数，其反卷积过程表示为：
其中和分别待重构样品散射势的最终反卷积频谱和初步合成频谱。Hsyn为合成孔径处理后系统的三维非相干传递函数，是Hsyn的共轭形式，ε为 正则化参数。
系统的三维非相干传递函数Hsyn可表示为：
其中j是虚数单位，λ为自由空间中的照明波长，P(uT)代表物镜光瞳函数，即二维相干传递函数，理想情况下是一个半径为NAobj/λ的圆函数，由物镜数值孔径NAobj决定。u＝(uT,uz)是对应于r的空间频率坐标，uT＝(ux,uy)是二维空间频率坐标。S是照明光源的空间频率强度分布函数。
选择加入正则化参数，目的是反卷积过程中防止噪声的过度放大。经验表明，反卷积性能在很大程度上取决于正则化参数的选择，而强度堆栈的信噪比也会影响最终层析成像结果的质量。为了防止噪声的过度放大，保证折射率信号不变，可将干镜和油镜的正则化参数分别设置为0.1和0.25附近。
步骤4，采用非负约束和全变分正则化相结合的混合迭代约束算法对合成的散射势频谱中缺失锥信息进行计算填充。
具体实施过程为：基于样品的先验知识，在空域中，认为样品的折射率始终高于介质的折射率，并以梯度值作为优化目标函数，而在频域中，认定实验测得的频谱数据是真实有效的。通过在空域和频域同时施加约束，反复迭代，可以在一定程度上填充散射势频谱中的失锥信息，获得更真实的结果。
步骤5，对最终合成的三维散射势频谱进行三维傅里叶逆变换，恢复待测样品的三维折射率分布。
具体实施过程为：对步骤5中得到的三维散射势频谱进行傅里叶逆变换，得到三维散射势O(r)。样品散射势函数又可以表示为其中n(r)为样品的三维复折射率分布，k0＝2π/λ是自由空间中照明波长为λ时的波矢量，nm为样品周围介质折射率。根据式
就可以得到表示样品的复折射率信息，其实部代表样品的折射率，虚部代表样品 的吸收。
本发明同时采用了可编程LED阵列和电动位移台。其中LED阵列作为照明光源保证了照明模式的编程可控，用来提供所需的可变照明角度的准单色平面波照明。电动位移台通过软件控制，配合LED阵列和相机曝光之间的时序信号，用来提供样品纳米级的轴向位移，实现了样品在不同照明角度下的三维光强图像堆栈的四维数据采集。利用三维空间域Kramers-Kronig关系在对数强度谱上执行三维半空间滤波，可以在仅强度测量下实现散射场复相位(振幅和相位)的完整信息恢复。结合非干涉合成孔径技术，将不同照明角度下的一阶散射场在三维频谱中进行拼接合成，其成像分辨率达到非相干衍射极限，并且成像结果不受散斑和寄生干涉的影响。

Claims (8)

  1. 一种基于非干涉合成孔径的光强传输衍射层析显微成像方法，其特征在于，步骤如下：
    步骤1，采集不同照角度下的轴向离焦强度堆栈；
    步骤2：计算不同入射光照下的三维对数强度谱，并对每个对数强度谱进行三维半空间傅里叶滤波，获得不同入射光照下包含复相位函数实部和虚部的三维散射场，在傅里叶空间合成所有单边带三维散射场，实现非干涉合成孔径，得到样品三维散射势频谱的初步估计；
    步骤3：对初步估计的频谱进行基于LED离散采样、照明部分相干和修正因子的三维反卷积；
    步骤4：采用非负约束和全变分正则化相结合的混合迭代约束算法对合成的散射势频谱中缺失锥信息进行计算填充；
    步骤5：对填充后的的三维散射势频谱进行三维傅里叶逆变换，恢复样品的三维折射率分布，实现无标记生物样品实现非侵入式三维成像。
  2. 根据权利要求1所述的基于非干涉合成孔径的光强传输衍射层析显微成像方法，其特征在于，利用基于非干涉合成孔径的光强传输衍射层析显微成像平台采集不同照角度下的轴向离焦强度堆栈，所述光强传输衍射层析显微成像平台包括包括可编程LED阵列、电动位移台扫描装置，待测样品、显微物镜、成像筒镜和相机，可编程LED阵列的圆心和显微物镜光轴重合，被放置在距离样品设置距离的位置，显微物镜的后焦面与筒镜的前焦面重合，相机的成像平面放置在成像筒镜的后焦面位置，成像时样品被放置在电动位移台上，逐个点亮LED单元，准单色平面波照射待测样品，通过物镜，经过成像筒镜汇聚后落在相机的成像平面上，通过控制电动位移台的轴向扫描，利用相机来记录三维光强堆栈。
  3. 根据权利要求1所述的基于非干涉合成孔径的光强传输衍射层析显微成像方法，其特征在于，步骤2中对不同入射光照下的三维强度堆栈取对数，并进行三维傅里叶变换，得到三维对数强度谱。
  4. 根据权利要求1或3所述的基于非干涉合成孔径的光强传输衍射层析显微成像方法，其特征在于，采用散射势函数O(r)来表征样品的三维结构，将散射势函数O(r)展开成实部和虚部的形式，即O(r)＝a(r)+jφ(r)，其中φ(r)和a(r)对应于散射势O(r)的相位成分和吸收成分；
    对不同入射光照下的三维强度堆栈I(r)取对数，表示为：
    式中，φ(r)和a(r)对应于散射势O(r)的相位和吸收，g(r)和g′(r)分别为层析成像系统的点扩散函数和对应的受入射光Uin(r)调制后的点扩散函数，g′*(r)是g′(r)的共轭形式；
    通过计算上式的傅里叶变换，得到对数强度谱函数：
    式中的和分别对应于三维强度堆栈I(r)、散射势O(r)的吸收成分a(r)和相位成分φ(r)的三维傅里叶变换，Ha(u)和Hφ(u)为衍射层析成像系统的吸收和相位传递函数。
  5. 根据权利要求4所述的基于非干涉合成孔径的光强传输衍射层析显微成像方法，其特征在于，衍射层析成像系统的吸收和相位传递函数分别表示为：
    式中，为系统的广义相干传递函数，u＝(uT,uz)是对应于r的空间频率坐标，um＝nm/λ，nm为样品周围介质折射率，λ为自由空间中的照明波长，P*(u)是P(u)的共轭形式，P(u+uin)和P*(u-uin)分别为P(u)和P*(u)受入射光空间频率uin平移调制后的相干传递函数表达式。
  6. 根据权利要求4所述的基于非干涉合成孔径的光强传输衍射层析显微成像方法，其特征在于，对每个对数强度谱进行三维半空间傅里叶滤波或三维希尔伯特变换，获得不同入射光照下包含复相位函数实部和虚部的三维散射场，在傅里叶空间合成所有单边带三维散射场，实现非干涉合成孔径，得到样品三维散射势频谱的初步估计的具体过程为：
    根据频谱中两个反对称广义孔径的位置，对每个双边带三维光谱进行三维半空间傅里叶滤波或三维希尔伯特变换，得到不同入射光照下包含复相位函数实部和虚部的三维散射场Us1(r)，根据傅里叶衍射定理
    在傅里叶空间合成所有单边带三维散射场，实现非干涉合成孔径，得到物体三维散射势频谱的初步估计，式中u＝(uT,uz)是对应于r的空间频率坐标，j是虚数单位，和分别对应于O和Us1的傅里叶变换，是受入射光空间频率uin平移调制后的散射势频谱，为系统的广义相干传递函数，其有限支持域被称为Ewald球壳。
  7. 根据权利要求1所述的基于非干涉合成孔径的光强传输衍射层析显微成像方法，其特征在于，步骤3中反卷积过程表示为：
    其中和分别样品散射势的最终反卷积频谱和初步合成频谱，Hsyn为合成孔径处理后系统的三维非相干传递函数，是Hsyn的共轭形式，ε为正则化参数。
  8. 根据权利要求7所述的基于非干涉合成孔径的光强传输衍射层析显微成像方法，其特征在于，合成孔径处理后系统的三维非相干传递函数具体为：
    其中j是虚数单位，λ为自由空间中的照明波长，P(uT)代表物镜光瞳函数，即二维相干传递函数，理想情况下是一个半径为NAobj/λ的圆函数，由物镜数值孔径NAobj决定，u＝(uT,uz)是对应于r的空间频率坐标，uT＝(ux,uy)是二维空间频率坐标，S是照明光源的空间频率强度分布函数。
PCT/CN2023/090075 2022-05-19 2023-04-23 一种基于非干涉合成孔径的光强传输衍射层析显微成像方法 WO2023221741A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210545129.5A CN114965470B (zh) 2022-05-19 2022-05-19 一种基于非干涉合成孔径的光强传输衍射层析显微成像方法
CN202210545129.5 2022-05-19

Publications (1)

Publication Number Publication Date
WO2023221741A1 true WO2023221741A1 (zh) 2023-11-23

Family

ID=82984409

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/090075 WO2023221741A1 (zh) 2022-05-19 2023-04-23 一种基于非干涉合成孔径的光强传输衍射层析显微成像方法

Country Status (2)

Country Link
CN (1) CN114965470B (zh)
WO (1) WO2023221741A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114965470B (zh) * 2022-05-19 2024-05-17 南京理工大学 一种基于非干涉合成孔径的光强传输衍射层析显微成像方法
CN115711866B (zh) * 2022-10-31 2024-07-09 西安电子科技大学 基于环形扫描照明的定量相衬层析显微装置和方法
CN116659414B (zh) * 2023-07-21 2023-10-13 南京信息工程大学 一种基于改进HiIbert变换的结构光解调方法
CN117268288B (zh) * 2023-07-26 2024-03-08 北京大学长三角光电科学研究院 光学衍射层析成像激光扫描方法、装置和电子设备
CN117314915B (zh) * 2023-11-29 2024-02-23 湖南大学 微结构加工的原位检测方法、设备及存储介质

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130057869A1 (en) * 2010-03-28 2013-03-07 Ecole Polytechnique Federale De Lausanne Complex index refraction tomography with sub lambda/6-resolution
US20140050382A1 (en) * 2011-01-24 2014-02-20 The Board Of Trustees Of The University Of Illinois Computational Adaptive Optics for Interferometric Synthetic Aperture Microscopy and Other Interferometric Imaging
US20140347672A1 (en) * 2011-07-29 2014-11-27 Ecole Polytechnique Federale De Lausanne (Epfl) Apparatus and method for quantitive phase tomography through linear scanning with coherent and non-coherent detection
CN108169173A (zh) * 2017-12-29 2018-06-15 南京理工大学 一种大视场高分辨三维衍射层析显微成像方法
CN109580457A (zh) * 2018-11-01 2019-04-05 南京理工大学 基于led阵列编码照明的三维衍射层析显微成像方法
CN111650738A (zh) * 2020-05-07 2020-09-11 清华大学深圳国际研究生院 一种基于深度学习的傅里叶叠层显微图像重构方法及装置
CN114965470A (zh) * 2022-05-19 2022-08-30 南京理工大学 一种基于非干涉合成孔径的光强传输衍射层析显微成像方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108362643B (zh) * 2018-02-10 2020-06-16 北京工业大学 一种双高度照明傅里叶叠层显微成像方法
CN111694016B (zh) * 2020-06-02 2023-10-20 南京理工大学 一种非干涉合成孔径超分辨成像重构方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130057869A1 (en) * 2010-03-28 2013-03-07 Ecole Polytechnique Federale De Lausanne Complex index refraction tomography with sub lambda/6-resolution
US20140050382A1 (en) * 2011-01-24 2014-02-20 The Board Of Trustees Of The University Of Illinois Computational Adaptive Optics for Interferometric Synthetic Aperture Microscopy and Other Interferometric Imaging
US20140347672A1 (en) * 2011-07-29 2014-11-27 Ecole Polytechnique Federale De Lausanne (Epfl) Apparatus and method for quantitive phase tomography through linear scanning with coherent and non-coherent detection
CN108169173A (zh) * 2017-12-29 2018-06-15 南京理工大学 一种大视场高分辨三维衍射层析显微成像方法
CN109580457A (zh) * 2018-11-01 2019-04-05 南京理工大学 基于led阵列编码照明的三维衍射层析显微成像方法
US20210372916A1 (en) * 2018-11-01 2021-12-02 Nanjing University Of Science And Technology 3d diffraction tomography microscopy imaging method based on led array coded illumination
CN111650738A (zh) * 2020-05-07 2020-09-11 清华大学深圳国际研究生院 一种基于深度学习的傅里叶叠层显微图像重构方法及装置
CN114965470A (zh) * 2022-05-19 2022-08-30 南京理工大学 一种基于非干涉合成孔径的光强传输衍射层析显微成像方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI JIAJI, ZHOU NING, SUN JIASONG, ZHOU SHUN, BAI ZHIDONG, LU LINPENG, CHEN QIAN, ZUO CHAO: "Transport of intensity diffraction tomography with non-interferometric synthetic aperture for three-dimensional label-free microscopy", LIGHT: SCIENCE & APPLICATIONS, NATURE PUBLISHING GROUP, UK, vol. 11, no. 1, UK , XP093110076, ISSN: 2047-7538, DOI: 10.1038/s41377-022-00815-7 *
ZUO CHAO; CHEN QIAN; SUN JIASONG; ANAND ASUNDI: "Non-Interferometric Phase Retrieval and Quantitative Phase Microscopy Based on Transport of Intensity Equation: A Review", CHINESE JOURNAL OF LASERS, SHANGHAI : SHANGHAI KEXUE JISHU CHUBANSHE, 1983-1992, CN, vol. 43, no. 06, 10 June 2016 (2016-06-10), CN , pages 227 - 257, XP009550678, ISSN: 0258-7025, DOI: 10.3788/CJL201643.0609002 *

Also Published As

Publication number Publication date
CN114965470B (zh) 2024-05-17
CN114965470A (zh) 2022-08-30

Similar Documents

Publication Publication Date Title
WO2023221741A1 (zh) 一种基于非干涉合成孔径的光强传输衍射层析显微成像方法
Park et al. Quantitative phase imaging in biomedicine
US11781966B2 (en) 3D diffraction tomography microscopy imaging method based on LED array coded illumination
Li et al. Transport of intensity diffraction tomography with non-interferometric synthetic aperture for three-dimensional label-free microscopy
US9546952B2 (en) Distribution of refractive index measurement by synthetic aperture tomography
JP6416887B2 (ja) 構造化照明を用いた組織試料の顕微鏡観察
US9185357B2 (en) Optical tissue sectioning using full field optical coherence tomography
CN107490562B (zh) 利用波面整形器的超高速三维折射率影像拍摄和荧光结构光照明显微镜系统及其使用方法
JP2016029388A (ja) Ｈｉｌｂｅｒｔ位相画像処理のためのシステムと方法
CN105182514A (zh) 基于led光源的无透镜显微镜及其图像重构方法
CN113317784A (zh) 一种微米级线式聚焦扫描显微光谱光学相干层析成像系统
CN114324245B (zh) 基于部分相干结构光照明的定量相位显微装置和方法
JP5002604B2 (ja) 偏光位相顕微鏡
Qiu et al. Spectral imaging with scattered light: from early cancer detection to cell biology
Sung Hyperspectral Three-Dimensional Refractive-Index Imaging Using Snapshot Optical Tomography
Tayebi et al. Real-time triple field of view interferometry for scan-free monitoring of multiple objects
Kang et al. Reflection-mode optical diffraction tomography for label-free imaging of thick biological specimens
CN105699332B (zh) 基于强度调制的白光干涉相位显微系统及其相位计算方法
CN116337811A (zh) 基于对向照明的光学衍射层析显微成像方法
CN207067518U (zh) 一种基于全息再现法实现照明的暗场数字显微装置
Tang Label-free Investigation of Cells with Phase Imaging, Diffraction Tomography, and Raman Spectroscopy
Ford Fast widefield techniques for fluorescence and phase endomicroscopy
CN118443624A (zh) 高速高分辨率三维衍射层析成像方法
Goswami et al. Quantitative phase imaging techniques for measuring scattering properties of cells and tissues: a review–part II
CN116256880A (zh) 一种基于干涉散射图像的漂移校正系统及其控制系统

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23806694

Country of ref document: EP

Kind code of ref document: A1