Lensless single-frame phase retrieval method based on partially coherent light-emitting diode illumination

Abstract

The application discloses a lensless single-frame phase recovery method based on partial coherent light emitting diode illumination, and the method is used on a lensless on-chip microscope system, only a monochromatic patch LED light source is used for illumination, and a single on-axis hologram is collected. Then, the high-resolution high-signal-to-noise ratio phase information of the object is directly recovered through a multi-wavelength iterative phase recovery method combined with dynamic phase support constraints. The application does not need to make complex transformation on the traditional lensless on-chip microscope, and can give the lensless on-chip microscope the ability of fast long-time single-frame phase recovery.

Description

Technical Field

[0001] This invention belongs to the field of optical microscopy measurement and markerless phase recovery technology, specifically a lensless single-frame phase recovery method based on partially coherent light-emitting diode illumination. Background Technology

[0002] In fields such as cell biology, digital pathology, and high-throughput drug screening, the demand for observing and recording subcellular information of high-throughput biological samples is increasing daily. However, traditional microscopes struggle to balance objective magnification with imaging field of view (FOV), making it impossible to simultaneously achieve high resolution and a large field of view. While high-precision scanning displacement platforms and stitching algorithms (Brown M, Lowe D G. Automatic panoramic image stitching using invariant features[J]. International journal of computer vision,2007,74(1):59-73.) can overcome these limitations, they also significantly increase the complexity of the system, making it difficult to widely apply in biological and medical disciplines.

[0003] In recent years, the emergence and development of lensless on-sheet holographic microscopy (LFOCM) has broken the limitations of field of view (FOV) and resolution in traditional microscopes. Its unique advantage lies in its ability to image directly on the original field of view of the imaging sensor without requiring any imaging lenses or other intermediate optical elements. The resolution of traditional lensless imaging systems is limited by pixel sampling frequency and diffraction limit. Due to its effective numerical aperture (NA) being close to 1, sensor pixel size is the main factor affecting imaging resolution. Therefore, in most lensless imaging systems, highly coherent laser sources and small-pixel sensors have been used to obtain high-resolution results. Meanwhile, in the field of "pixel super-resolution," many methods have been invented, such as active parallel plate scanning, axial scanning of the sample-to-sensor distance, or multi-wavelength scanning using ultra-wide lasers, to overcome the limitations of sensor pixel sampling frequency and achieve even higher resolution.

[0004] However, in the field of optical phase microscopy, the use of partially coherent illumination is of great significance for improving imaging quality and resolution and suppressing coherent noise. Therefore, in recent years, there have been many related works at home and abroad. Ozcan et al. developed a miniaturized lensless system using LED illumination (Pushkarsky, I., Liu, Y., Weaver, W. et al. Automated single-cell motility analysis on a chip using lensfree microscopy. Sci Rep4, 4717 (2014). https: / / doi.org / 10.1038 / srep04717). Zuo et al. used LED array illumination to achieve mechanical displacement-free experimental system (Zuo C, Sun J, Zhang J, et al. Lensless phase microscopy and diffraction tomography with multi-angle and multi-wavelength illuminations using an LED matrix[J]. Optics express, 2015, 23(11): 14314-14328.). Compared with laser light sources, LEDs can reduce coherent noise, improve imaging quality and simplify the entire imaging system. However, according to previous theoretical analyses by Zuo et al. (Zhang J, Sun J, Chen Q, et al. Resolution analysis in a lens-free on-chip digital holographic microscope[J]. IEEE Transactions on Computational Imaging, 2020, 6: 697-710.), insufficient spatiotemporal coherence of LED light sources leads to the mixing of high-frequency diffraction information into the low-frequency region, resulting in additional resolution loss, and even replacing the pixel sampling frequency as the main factor limiting imaging resolution.

[0005] To address the aforementioned issues and achieve higher resolution in partially coherent illumination systems, only a few studies have so far compensated for the resolution loss caused by insufficient spatiotemporal coherence of partially coherent light sources. Ozcan et al. used coherent mode decomposition to compensate for the impact of temporal coherence on imaging resolution (Sencan I, Coskun AF, Sikora U, et al. Spectral demultiplexing in holographic and fluorescent on-chip microscopy[J]. Scientific reports, 2014, 4(1): 1-9.), while Feng et al. used deconvolution to compensate for the resolution loss caused by insufficient spatial coherence to some extent (Feng S, Wu J. Resolution enhancement method for lensless in-line holographic microscope with spatially-extended light source[J]. Optics express, 2017, 25(20): 24735-24744.). However, in the above methods, they only image sparse samples or simple resolution lines. Therefore, to date, there have been no reports on single-frame phase retrieval techniques for lensless on-sheet microscopes based on LED illumination, nor on long-term dynamic quantitative phase imaging observations of biological samples. Summary of the Invention

[0006] The purpose of this invention is to provide a lensless single-frame phase recovery method based on partially coherent light-emitting diode illumination.

[0007] The technical solution to achieve the objective of this invention is as follows: a lensless single-frame phase retrieval method based on partially coherent light-emitting diode illumination, comprising the following steps:

[0008] Step 1: Acquire a holographic image of the sample;

[0009] Step 2: Segment the LED broadband wavelengths according to spectral priors and initialize phase constraint information;

[0010] Step 3 involves updating the complex amplitude of the object using the digitally segmented sub-wavelengths and the captured hologram to obtain the object's phase. This specifically includes:

[0011] By utilizing the segmented sub-wavelengths of a broadband LED, the original intensity image is propagated to the focal plane to obtain the complex amplitude of the object surface, and phase support constraints and uniform light intensity constraints are applied to the phase and amplitude of the complex amplitude, respectively.

[0012] The updated complex amplitude is propagated back to the camera surface using angular spectrum theory to obtain the phase information of the current segmented sub-wavelength propagated back to the camera plane. The camera surface amplitude information is updated based on the captured hologram and spectral prior weight coefficients. All segmented sub-wavelengths are traversed and the phase information is recovered.

[0013] Preferably, a lensless on-plate microscopy system is used to acquire holographic images. The lensless on-plate microscopy system includes a single light-emitting diode (LED) illumination source and a sensor. The LED illumination source is a broadband, non-monochromatic, patch LED illumination source.

[0014] Preferably, step 2 specifically includes:

[0015] Based on the prior knowledge of the spectrum of the lighting LED, the lighting wavelength λ is divided into N equally spaced sub-wavelengths {λ...} i ,i=1,2,...,N};

[0016] Using the center wavelength λ c The captured light intensity map I ini The complex amplitude of the camera surface is generated by combining the initial zero phase ψ0=0. The complex amplitude is propagated back to the object plane, and then the complex amplitude of the object plane is used. The phase portion is used to obtain the initial phase constraint Mask0, which is dynamically updated to Mask in subsequent iterations. i .

[0017] Preferably, the specific steps for updating and iterating the complex amplitude of the object using digitally segmented sub-wavelengths and captured holograms to obtain the object's phase are as follows:

[0018] Step 3.1, generate the light intensity map I. ini Combined with the previous sub-wavelength λ i-1 The phase information propagated back to the camera plane generates the complex amplitude of the camera plane;

[0019] Step 3.2: Propagate the complex amplitude to the object surface at the corresponding sub-wavelength to obtain the object surface complex amplitude;

[0020] Step 3.3: Apply phase support constraints to the phase portion of the complex amplitude of the object surface, update the phase according to the segmented sub-wavelengths, and simultaneously apply uniform light intensity constraints to the amplitude portion. Where ave(·) is the mean calculation operator, which obtains the updated complex amplitude.

[0021] Step 3.4, update the complex amplitude of the object surface. Propagation back to the camera plane yields the segmented sub-wavelength λ. i The phase information is propagated back to the camera plane, and the process returns to step 3.1, combining it with the next segmented sub-wavelength λ. i+1The camera surface complex amplitude is updated with weights until all segmented sub-wavelengths are traversed, thereby obtaining the high resolution and high signal-to-noise ratio phase ψ of the sample.

[0022] Preferably, the camera surface complex amplitude generated in step 3.1 is specifically as follows:

[0023]

[0024] In the formula, Let ω be the complex amplitude of the camera plane. i The weight of the i-th segmented sub-wavelength, j is the imaginary unit, ψ i For the previous sub-wavelength λ i-1 Phase information is propagated back to the camera plane.

[0025] Preferably, the complex amplitude of the object surface obtained in step 3.2 is specifically as follows:

[0026]

[0027] In the formula, H(-d) is the transfer function for the propagation distance -d. The complex amplitude of the object's surface. For the above sub-wavelength λ i-1 The mode of the complex amplitude after propagation back to the object plane, The phase of the object surface.

[0028] Compared with the prior art, the present invention has the following significant advantages: (1) It can obtain the corresponding resolution results under laser illumination while using patch LED illumination to simplify the imaging system. (2) It only requires taking a single hologram without any displacement platform or stitching algorithm. The system is more compact and cost-effective, and can perform real-time long-term imaging of live cells in an incubator with an ultra-large field of view.

[0029] The present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the experimental setup in a lensless single-frame phase retrieval method based on partially coherent LED lighting.

[0031] Figure 2 This is a flowchart of a lensless single-frame phase recovery method based on partially coherent LED lighting.

[0032] Figure 3 It consists of the captured single-frame hologram, the phase result directly transmitted back for reconstruction, and the final reconstructed phase resolution plate result.

[0033] Figure 4It is a long-term live-cell phase result reconstructed using the present invention and a digital multimodal result achieved without the use of any external devices. Detailed Implementation

[0034] like Figure 2 As shown, a lensless single-frame phase retrieval method based on partially coherent light-emitting diode illumination includes the following four steps:

[0035] Step 1: Acquire holographic images using a lensless on-plate microscopy system.

[0036] like Figure 1 As shown, this invention is based on a traditional lensless on-sheet microscopy system structure, comprising only a light source 1, a sample 2, and a sensor 3. The monochromatic patch LED illumination source 1 used in this invention has a 632nm wavelength, a bandwidth of 23.95nm, and a light source area of ​​0.0091mm². 2 The sample was directly illuminated. Using the measured spectral width and experimental verification, the spectral width of the light source was digitally divided into 20 quasi-monochromatic sub-wavelengths with 2nm intervals for sample reconstruction. Sample 2 was placed directly on the image sensor for imaging. This system uses a board-level monochromatic CMOS sensor 3 (pixel size 0.9μm, 5664×4256, Jiangsu Teamone Intelligence Technology Co., Ltd.).

[0037] The specific implementation process is as follows: a broadband LED incoherent light source is used to directly illuminate the sample, while simultaneously triggering a camera to record a holographic image at the center wavelength. ini .

[0038] Step 2: Segment the broadband wavelength of the LED according to the spectral prior and initialize the phase constraint information.

[0039] The specific implementation process is as follows: Based on the prior spectrum of the lighting LED, the lighting wavelength λ is divided into N equally spaced sub-wavelengths {λ...} i ,i=1,2,...,N};

[0040] Using the center wavelength λ c The captured light intensity map I ini The complex amplitude of the camera surface is generated by combining the initial zero phase ψ0=0. The complex amplitude is propagated back to the object plane, and then the complex amplitude of the object plane is used. The phase portion is used to obtain the initial phase constraint Mask0, which is dynamically updated to Mask in subsequent iterations. i .

[0041] Step 3: Update the complex amplitude of the object using digitally segmented sub-wavelengths and the captured hologram. The specific implementation process is as follows:

[0042] Step 3.1, place hologram I ini Combined with the previous sub-wavelength λ i-1 The phase information propagated back to the camera plane generates the complex amplitude of the camera plane, and the specific formula is as follows:

[0043]

[0044] In the formula, Let ω be the complex amplitude of the camera plane. i The weight of the i-th segmented sub-wavelength, j is the imaginary unit, ψ i For the previous sub-wavelength λ i-1 Phase information propagated back to the camera plane (initially 0).

[0045] Step 3.2: Propagate the complex amplitude to the object surface at the corresponding sub-wavelength to obtain the complex amplitude of the object surface. The specific formula is as follows:

[0046]

[0047] In the formula, H(-d) is the transfer function for the propagation distance -d. The complex amplitude of the object's surface. For the above sub-wavelength λ i-1 The mode of the complex amplitude after propagation back to the object plane, The phase of the object surface.

[0048] Step 3.3: Apply phase support constraints to the phase portion of the complex amplitude of the object surface. And update the phase according to the segmented wavelength. At the same time, a uniform light intensity constraint is applied to the amplitude part. Here, ave(·) is the mean calculation operator, which yields the updated complex amplitude.

[0049] Step 3.4, update the complex amplitude of the object surface. Propagation back to the camera plane yields the segmented sub-wavelength λ. i The phase information is propagated back to the camera plane, and the process returns to step 3.1, combining it with the next segmented sub-wavelength λ. i+1 The camera surface complex amplitude is updated with weights until all segmented sub-wavelengths are traversed, thereby obtaining the high resolution and high signal-to-noise ratio phase ψ of the sample.

[0050] Figure 3 The captured holographic pattern is shown, and compared with the resolution of the traditional single-frame LED method (1.381 μm), it demonstrates that the present invention achieves a resolution improvement of 1.41 times (0.977). Figure 4 The present invention utilizes the reconstructed dynamic results of long-term cultured live cells and the phase contrast, differential symmetry, and three-dimensional structural images reconstructed by digital methods.

[0051] This invention requires only vertical illumination from a light source using a patch-mounted broadband LED to obtain a coaxial hologram. Then, a multi-wavelength phase retrieval and reconstruction algorithm combined with a dynamic phase support constraint method, a propagation model, and an incoherent superposition model are used to continuously propagate and update this intensity image at different segmented sub-wavelengths in the camera and object planes, gradually recovering the sample's phase information. This invention uses only a single patch-mounted LED light source and introduces no mechanical displacement, improving system stability and significantly reducing costs.

Claims

1. A lensless single-frame phase retrieval method based on partially coherent light-emitting diode illumination, characterized in that, The steps are as follows: Step 1: Acquire a holographic image of the sample; Step 2: Segment the LED broadband wavelength according to spectral priors and initialize phase constraint information, including: Based on the prior knowledge of the spectrum of the lighting LED, the lighting wavelength Divided into N equally spaced sub-wavelengths ; Utilizing the center wavelength The captured light intensity map Combined with the initial zero phase Generate camera surface complex amplitude The complex amplitude is propagated back to the object plane, and then the complex amplitude of the object plane is used. The phase portion obtains initial phase constraints. Initial constraints Dynamically updated in subsequent iterations ; Step 3 involves updating the complex amplitude of the object using digitally segmented sub-wavelengths and the captured hologram to obtain the object's phase. This specifically includes: By utilizing the segmented sub-wavelengths of a broadband LED, the original intensity image is propagated to the focal plane to obtain the complex amplitude of the object surface. Phase support constraints and uniform intensity constraints are then applied to both the phase and amplitude of the complex amplitude. Specifically, a phase support constraint is applied to the phase portion of the complex amplitude of the object surface. And update the phase according to the segmented sub-wavelength. ; The updated complex amplitude is propagated back to the camera surface using angular spectrum theory to obtain the phase information of the current segmented sub-wavelength propagated back to the camera plane. The camera surface amplitude information is updated based on the captured hologram and spectral prior weight coefficients. All segmented sub-wavelengths are traversed and the phase information is recovered.

2. The lensless single-frame phase retrieval method based on partially coherent LED illumination according to claim 1, characterized in that, Holographic images are acquired using a lensless on-chip microscopy system, which includes a single light-emitting diode (LED) illumination source and a sensor. The LED illumination source is a broadband, non-monochromatic, surface-mount LED illumination source.

3. The lensless single-frame phase retrieval method based on partially coherent LED illumination according to claim 1, characterized in that, The specific steps are as follows: Step 3.1, obtain the light intensity map Combined with the previous segmented wavelength The phase information propagated back to the camera plane generates the complex amplitude of the camera plane; Step 3.2: Propagate the complex amplitude to the object surface at the corresponding segmented wavelength to obtain the object surface complex amplitude; Step 3.3: Apply phase support constraints to the phase portion of the complex amplitude of the object surface, update the phase according to the segmented sub-wavelengths, and simultaneously apply uniform light intensity constraints to the amplitude portion. ,in The mean-calculation operator is used to obtain the updated complex amplitude. ; Step 3.4, update the complex amplitude of the object surface. Propagate back to the camera plane to obtain the segmented sub-wavelengths. The phase information propagated back to the camera plane is returned to step 3.1, and combined with the next segmented sub-wavelength. The camera's complex amplitude is updated with weights until all segmented sub-wavelengths are traversed, thereby obtaining a high-resolution and high signal-to-noise ratio phase of the sample. .

4. The lensless single-frame phase retrieval method based on partially coherent LED illumination according to claim 3, characterized in that, The camera surface complex amplitude generated in step 3.1 is as follows: ， In the formula, The complex amplitude of the camera plane. The weight of the i-th segmented sub-wavelength, The imaginary unit, For the previous sub-wavelength Phase information is propagated back to the camera plane.

5. The lensless single-frame phase retrieval method based on partially coherent LED illumination according to claim 3, characterized in that, The complex amplitude of the object surface obtained in step 3.2 is as follows: ， In the formula, For the distance of transmission The transfer function, The complex amplitude of the object's surface. For the above-mentioned sub-wavelength The mode of the complex amplitude after propagation back to the object plane, The phase of the object surface.

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