Array polarization type three-dimensional living cell high-resolution holographic dynamic microscopic imaging system

Abstract

The invention discloses an array polarization type three-dimensional living cell high-resolution holographic dynamic microscopic imaging system, belongs to the technical field of optical microscopic imaging, and aims to solve the problems that the imaging flux is limited, the space resolution and the time resolution are mutually restricted and the like in the current three-dimensional living cell microscopic imaging system. According to the system, a low-power objective lens is matched with an array type white light source to design an illumination module, the spatial resolution of the system is optically enhanced by a coding illumination technology, and high-flux optical imaging under the conditions of a wide view field and high resolution in a spatial dimension is realized; a polarization interference modulation light path is designed by matching an optical polarization device with a spatial light modulator (SLM), the time resolution capability of the system is enhanced by a digital holographic technology, and time-dimensional fast phase imaging is realized. Compared with the existing three-dimensional living cell microscopic imaging system, the method disclosed by the invention has the characteristics of non-contact type, no mark, high flux, long-time dynamic microscopic imaging and the like, and has certain advantages in the aspects of system complexity, cost and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical microscopic imaging, and particularly relates to an array polarization type three-dimensional live cell high-resolution holographic dynamic microscopic imaging system. Background Art

[0002] Three-dimensional optical microscopic imaging of live cells can collect and provide three-dimensional spatial and temporal information of dynamic events occurring in cell tissues, single cells, intracellular networks (formerly), and even organisms (in vivo), and is a necessary technology for studying dynamic physiological processes in disciplines such as cell biology, cancer science, developmental biology, and neuroscience. In recent years, with the rapid development of disciplines such as electronics, optics, materials science, biochemistry, and computer technology, three-dimensional optical microscopic imaging of live cells has quickly become a research hotspot in the interdisciplinary field, attracting extensive research and attention from many well-known scientific research teams at home and abroad, and achieving a series of important innovative results.

[0003] To achieve precise observation at the level between live cells and within cells, researchers have proposed a series of far-field super-resolution microscopic imaging methods based on fluorescence microscopy. In recent years, the Ralf team at the Max Planck Institute of Biochemistry in Germany proposed the DNA barcode method of Exchange-PAINT to record photon scintillation events required in single molecule microscopic imaging, and further improved the system resolution through the sequential imaging method (RESI), achieving an angstrom-scale spatial resolution on the existing fluorescence microscopy system. However, current super-resolution microscopic techniques often require complex acquisition equipment and specific imaging control elements, and they often pursue high-resolution capabilities in the spatial dimension at the expense of the resolution in the time dimension.

[0004] In the research on cell cancer evolution and medical treatment, Raman scattering and Brillouin scattering are highly sensitive ways to detect the chemical composition and mechanical properties of cells. Compared with traditional slicing methods, these two methods have obvious advantages such as non-contact, non-destructive, and high-resolution. In recent years, Professor Jitao Zhang of Wayne State University and the Giuliano research group at the University of Maryland have cooperated to develop a Brillouin microscope based on dual-line scanning, achieving an acquisition speed 50-100 times faster than the existing technology and reducing the phototoxicity by an order of magnitude. However, factors such as the system cost, system complexity, total length, and total weight of the current fluorescence microscopy-based high-throughput imaging system are still difficult problems restricting the practical application of imaging instruments. Moreover, fluorescence imaging phototoxicity is also an important factor restricting long-term imaging of live cells.

[0005] On the other hand, quantitative phase imaging (QPI) has gradually become a research hotspot in live cell imaging. Compared with fluorescence imaging methods, quantitative phase imaging is an ideal observation method for three-dimensional label-free and non-invasive imaging of transparent live cell samples. However, traditional quantitative phase imaging is represented by implementation techniques such as laminar microscopy, digital holography, spatial light interference microscopy, and intensity transport equation. These techniques each have unique and mature imaging principles, system structures, and performance advantages, and it is difficult to combine them with each other to complement their advantages and form an optical imaging method that takes into account both spatial and temporal resolution capabilities. In recent years, the application mode combining deep learning technology has also gradually become a research hotspot in live cell imaging. Deep learning technology has many advantages such as stronger robustness, lower computational complexity, and faster processing speed. The combination of the two can effectively reduce the computational cost and system cost. Based on digital holography and deep learning, the Aydogan Ozcan team at the University of California, Los Angeles, developed a low-cost and compact stain-free imaging device

[29] , which is suitable for virology research, vaccine development, and clinical diagnosis. Based on this imaging device, the researchers achieved a virus concentration measurement detection result that is 10 times larger in dynamic range than the gold standard method and is more efficient and immediate. However, the imaging method based on deep learning always faces two important influencing factors - data volume and versatility, which limit its wide application in related fields. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an array polarization three-dimensional live cell high-resolution holographic dynamic microscopy imaging system. By using the designed white light encoding and external single-light-path illumination conditions, and collocating polarization elements, it is ensured that the double-polarization illumination optical path is consistent with the background noise, providing a prerequisite for high-quality optical information acquisition of the sample. Then, the double-polarization light field undergoes the same multi-layer scattering effect in the sample and is affected by exactly the same degradation model. Therefore, the imaging optical system has three-dimensional optical sectioning tomography ability. Finally, using holographic optics and polarization optics elements, a fixed phase shift and coherent conditions are generated for the double-polarization light field, and the holographic pattern of the sample is collected on the surface of the detector.

[0007] An array polarization three-dimensional live cell high-resolution holographic dynamic microscopy imaging system, comprising a white light LED array light source (1), a polarizer (2), a first Wollaston prism (3), a second Wollaston prism (7), a beam splitter (10), a spatial light modulator SLM (11), an analyzer (12), and an imaging detector (14); The light beam emitted by the white light LED array light source (1) is first modulated by the polarizer (2) at a 45° angle to generate linearly polarized light in the 45° direction; subsequently, the light beam enters the first Wollaston prism (3), is converted into two mutually orthogonal linearly polarized lights, passes through the sample (5), and then passes through the second Wollaston prism (7) and enters the imaging detector (14); the light beam transmitted by the second Wollaston prism (7) irradiates on the reflective spatial light modulator SLM (11); the double polarized light is reflected by the spatial light modulator SLM (11), and then reflected by the beam splitter (10) and exits as parallel light into the second analyzer (12) in the 45° direction; the second linearly polarized light modulated by the analyzer (12) is transmitted to the imaging detector (14) and interferes with the first linearly polarized light; the imaging detector (14) generates a three-dimensional tomographic image through axial scanning.

[0008] Preferably, it further includes a condenser lens (4); the linearly double polarized light is converged by the condenser lens (4) and then passes through the sample (5).

[0009] Preferably, it further includes an objective lens (6); the double polarized light passes through the objective lens (6) and is focused on the second Wollaston prism (7) located at the rear focal plane.

[0010] Preferably, it further includes a first lens (9); the light beam transmitted by the second Wollaston prism (7) becomes parallel light through the converging effect of the first lens (9) and irradiates on the reflective spatial light modulator SLM (11).

[0011] Preferably, it further includes a second lens (13); the second linearly polarized light modulated by the analyzer (12) is focused on the imaging detector (14) through the second lens (13).

[0012] Preferably, the spatial light modulator SLM (11) is responsible for regulating the phase delay of a polarized light beam to generate an additional phase difference.

[0013] Preferably, the white light LED array light source (1) is a programmable white light LED array.

[0014] Preferably, the objective lens (6) is a low-magnification objective lens, which is responsible for providing imaging resolution and a wide imaging field of view.

[0015] Preferably, it further includes a tube lens (8); the tube lens (8) and the first lens (9) form an image-space telecentric optical path and are responsible for broadening the original polarized light beam into a parallel optical path with a wider range.

[0016] The present invention has the following beneficial effects: 1. Based on the holographic optical imaging principle, by flexibly integrating and applying polarization optical elements and stacked imaging optical paths, etc., while ensuring efficient and accurate imaging, the system complexity and total cost of the imaging optical system are reduced.

[0017] 2. By using white light array illumination and combining with coded illumination technology, the limitation condition between the spatial resolution ability of the objective lens of the optical system and the field of view can be broken, the gain bandwidth product of the system can be broadened, and the information throughput of the imaging system can be improved.

[0018] 3. By using the concept of digital holography and combining with a phase spatial light modulator (SLM), the phase information in the ptychography can be accurately and quickly solved, the pain point problem of long-time consumption in ptychography can be solved, and the imaging condition for dynamic microscopy can be provided.

[0019] 4. The imaging principle of this system belongs to the category of label-free and non-contact quantitative phase imaging, and can provide the imaging condition for long-time live cells. At the same time, the designed polarization imaging optical path provides a single-path interference condition and a consistent degradation model for the digital holographic method, enabling the system to have the ability of optical tomography and obtain three-dimensional imaging ability. Description of the Drawings

[0020] Figure 1 It is the schematic diagram of the microscopic imaging system of the present invention.

[0021] Among them, 1 - white light LED array, 2 - polarizer, 3 - first Wollaston prism, 4 - condenser, 5 - sample, 6 - objective lens, 7 - second Wollaston prism, 8 - tube lens, 9 - first lens, 10 - beam splitter, 11 - spatial light modulator SLM, 12 - analyzer, 13 - second lens, 14 - imaging detector. Detailed Embodiments

[0022] The following combines the drawings and gives embodiments to describe the present invention in detail.

[0023] The array polarization three-dimensional live cell high-resolution holographic dynamic microscopic imaging system of the present invention includes: white light LED array 1, polarizer 2, first Wollaston prism 3, condenser 4, objective lens 6, second Wollaston prism 7, tube lens 8, first lens 9, beam splitter 10, SLM 11, analyzer 12, second lens 13, imaging detector 14. The imaging principle of this system is described in the following three parts: 1. White light coded illumination. Different from the halogen light source of traditional optical imaging systems or the laser light source of holographic optical systems, this system uses a low-cost programmable white light LED array 1 as the illumination source. For an LED light source array 1 with M rows and N columns, it has a total of MN units that can be gated and coded. In one imaging, if the lit state of a single LED is regarded as 1 and the unlit state is regarded as 0, then the illumination coding state of the entire light source array 1 is recorded as a coding matrix containing only 0 and 1 elements. Conversely, in this way, the computer can conveniently control the illumination method of each imaging application by setting different coding matrices, which is the coded illumination technology. This system comprehensively considers the dynamic imaging requirements, mainly aims to code and cover the array units within the radius of a circle twice the numerical aperture (NA) of the objective lens, and realizes imaging illumination and expands the sample spectrum information range through four encodings.

[0024] 2. White light polarization modulation. Traditional digital holographic imaging often uses highly coherent monochromatic laser illumination to form stable interference conditions, but at the same time introduces problems such as laser artifacts. To avoid this problem, this system uses low-coherence light - white light as the illumination condition, and uses the principle of polarization beam splitting modulation to generate two polarized lights with almost the same optical path structure to provide stable holographic interference conditions for the follow-up.

[0025] When the light beam emitted by the white light LED array light source 1 is first modulated by the polarizer 2 in the 45° direction, linearly polarized light in the 45° direction is generated; subsequently, the light beam enters the first Wollaston prism 3 and is converted into two orthogonally polarized linearly polarized lights; the two polarized lights are converged by the condenser lens 4 and then pass through the sample 5, and the optical path difference required for interference is generated by modulation in adjacent regions of the sample space. However, the two polarized lights do not interfere with each other due to different polarization states; subsequently, the two polarized lights pass through the objective lens 6 and are focused on the second Wollaston prism 7 located on the rear focal plane and are converted into parallel light and emitted; Finally, the light beam is reflected by the beam splitter 10 to the second polarizer 12 in the 45° direction and is converted into linearly polarized light with the same direction, thus providing stable interference conditions. Since before passing through the second polarizer 12, the optical paths modulated by the sample do not produce interference effects due to different polarization states, and the optical path difference is only generated due to the thickness and refractive index of the sample itself. The two polarized lights have almost the same optical path structure (microscopic translation in the sample area), background noise and sample multiple scattering degradation effects. Therefore, white light illumination conditions can be used throughout the process before the imaging light beam enters the spatial light modulator SLM 11.

[0026] 3. Single-path polarization holography. This system uses an optical intensity and phase modulation element - a spatial light modulator (SLM) to achieve high-quality quantitative phase imaging.

[0027] The light beam emerging from the second Wollaston prism 7 becomes parallel light due to the converging effect of the first lens 9 and irradiates the reflective SLM 11. The SLM 11 is responsible for regulating the phase delay of a polarized light beam to generate a fixed additional phase difference, while the other polarized light beam remains unchanged. Subsequently, the two polarized light beams are reflected by the SLM 11, and then reflected by the beam splitter 10 and emerge as parallel light into the second polarizer 12 at a 45° direction. The parallel light modulated by the polarizer 12 enters the second lens 13 and is focused on the imaging detector 14 to generate interference, and the camera captures the holographic pattern containing the sample information. Finally, the imaging detector 14 performs axial scanning at a fixed interval through the Z-axis precision device to generate a three-dimensional tomographic image.

[0028] Different from the design of traditional digital holographic imaging systems, the above-mentioned polarization holographic process can be realized by only one optical path without setting up another precise reference optical path. To ensure the modulation effect, white light filtering treatment is also required with a narrow-band filter before the polarized light enters the SLM. On the other hand, the phase modulation method adopted in this system is a reflective SLM. Compared with traditional phase modulation elements and methods, the SLM has obvious advantages such as simplicity, rapidity, and precision, providing a strong guarantee for high-quality information acquisition and encoding of samples.

[0029] The principle of high-throughput, dynamic microscopic imaging of living cells by this array polarization three-dimensional living cell high-resolution holographic dynamic microscopy system is as follows: 1. High-throughput microscopic imaging. There is a negative correlation between the field of view and the resolution ability of the microscopic objective lens. Different from traditional holographic microscopy techniques that use high-magnification objective lenses for imaging, this system uses a low-magnification objective lens to ensure wide-field imaging conditions. At the same time, the equivalent numerical aperture of the optical system imaging is extended through white light coded illumination technology to improve the spatial resolution ability of the system, thereby realizing high-throughput imaging.

[0030] The principle of extending the numerical aperture of the optical system imaging is as follows: In the imaging stage, an LED light source array containing M rows and N columns is placed on the horizontal plane above the sample to provide oblique illumination conditions. Assume that the central wavelength of the light wave emitted by the LED is . Assume that the distance between the light source horizontal plane and the sample plane is far enough, and the distance between them is . Then the light wave emitted from a single LED unit in the array can be regarded as an ideal plane wave. If a single LED located in the th row and the th column of the array is lit alone, then the two-dimensional wave vector of its emitted light can be expressed as:

[0031] Among them, and respectively represent the two-dimensional spatial coordinates of the illuminated LED and the central LED in the array. For the sake of convenient description, let the amplitude of the initial incident light field be 1 and have a consistent phase of 0, and the spatial wave vector coordinates are denoted as , then the wave function of the incident light field can be denoted as .

[0032] Subsequently, the incident light field propagates through space to the sample plane, illuminates the sample, and exits from the sample plane, then the light field is modulated by the sample information. Let the complex amplitude in the spatial domain of the sample be (transmission modulation function), then in the Fourier domain, the sample spectrum function exiting from the sample can be expressed as:

[0033] where represents the complex amplitude function of the light field exiting from the sample, represents the spectrum function exiting from the sample under normal incidence conditions. Equation 2 shows that the modulation effect of the LED unit illumination wave vector on the sample spectrum is actually to translate the initial spectrum position of the sample from the center by units.

[0034] Subsequently, the light field exiting from the sample is focused by the objective lens and then propagates to the back focal plane. If an imaging detector is placed here (image plane), then the camera can collect the low-resolution intensity image of the sample . Let the frequency-domain pupil function of the objective lens be , combining Equation 2, the mathematical model of the above process can be expressed as:

[0035] where, is a frequency-domain low-pass filtering function determined by the numerical aperture of the objective lens. Equation 3 shows that the ptychography uses the spatial illumination wave vectors generated by LEDs at different positions in the light source array, so that the frequency-domain sampling sub-aperture is translated to , thus having the ability to collect high-throughput sample information beyond the original numerical aperture range of the objective lens.

[0036] 2. Live cell dynamic phase imaging. Non-contact, label-free quantitative phase imaging is an excellent way to achieve long-term live cell imaging.

[0037] To quickly calculate the spectrum information of each sub-aperture in ptychography and effectively avoid the speed disadvantage of the phase retrieval iterative algorithm in ptychography, this system uses a reflective SLM device to generate holograms by four-step phase-shift interference, so as to achieve quantitative phase imaging.

[0038] In the imaging stage, the parallel light emerging from the second Wollaston prism enters the reflective SLM. The reflective SLM introduces a phase delay to the light field of a single polarization state , and keeps the other polarization light field unchanged:

[0039] Subsequently, the modulated light beam enters the camera plane through the analyzer to generate coherent interference:

[0040] where, represents the spatial displacement difference between the light fields of the two polarization states, represents the imaging light field distribution of a single polarization state. Finally, the camera collects and records the interference hologram of the light field :

[0041] where, and represent the intensity and phase distributions of the imaging light field respectively. represents the mutual intensity distribution of the two spatially displaced light fields:

[0042] Based on the four collected holographic interference intensity maps ( ), the phase gradient distribution of the imaging light field can be directly solved :

[0043] Therefore, the phase information of the imaging light field of the sub-aperture can be directly obtained by integrating along the gradient direction. Taking the axis as an example, for a known reference point phase value , the phase distribution of the imaging light field is:

[0044] In summary, the above is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An array polarization type three-dimensional live cell high-resolution holographic dynamic microscopic imaging system, characterized in that, It includes a white light LED array light source (1), a polarizer (2), a first Wollaston prism (3), a second Wollaston prism (7), a beam splitter (10), a spatial light modulator SLM (11), an analyzer (12), and an imaging detector (14); The light beam emitted from the white light LED array light source (1) is first modulated by the polarizer (2) in the 45° direction to generate linearly polarized light in the 45° direction. Subsequently, the light beam enters the first Wollaston prism (3), is converted into two mutually orthogonal linearly polarized light beams, passes through the sample (5), then passes through the second Wollaston prism (7), and enters the imaging detector (14). The light beam transmitted through the second Wollaston prism (7) irradiates on the reflective spatial light modulator SLM (11). The double-polarized light is reflected by the spatial light modulator SLM (11), and then reflected by the beam splitter (10) and exits as parallel light into the second analyzer (12) in the 45° direction. The second linearly polarized light modulated by the analyzer (12) is transmitted to the imaging detector (14) and interferes with the first linearly polarized light. The imaging detector (14) generates a three-dimensional tomographic image through axial scanning.

2. The array polarization type three-dimensional live cell high-resolution holographic dynamic microscopic imaging system according to claim 1, wherein It further includes a condenser lens (4); the linearly double-polarized light is converged by the condenser lens (4) and then passes through the sample (5).

3. The array polarization type three-dimensional live cell high-resolution holographic dynamic microscopy imaging system according to claim 2, wherein It further includes an objective lens (6); the double-polarized light passes through the objective lens (6) and is focused on the second Wollaston prism (7) located at the rear focal plane.

4. The array polarization type three-dimensional live cell high-resolution holographic dynamic microscopic imaging system according to claim 3, characterized in that, It further includes a first lens (9); the light beam transmitted through the second Wollaston prism (7) becomes parallel light through the converging effect of the first lens (9) and irradiates on the reflective spatial light modulator SLM (11).

5. An array polarization type three-dimensional live cell high-resolution holographic dynamic microscopic imaging system according to claim 4, characterized in that, It further includes a second lens (13); the second linearly polarized light modulated by the analyzer (12) is focused on the imaging detector (14) by the second lens (13).

6. The three-dimensional live cell high-resolution holographic dynamic microscopy imaging system with array polarization as claimed in claim 1, wherein The spatial light modulator SLM (11) is responsible for regulating the phase delay of a polarized light beam to generate an additional phase difference.

7. An array polarization type three-dimensional live cell high-resolution holographic dynamic microscopy imaging system according to claim 1, characterized in that, The white light LED array light source (1) is a programmable white light LED array.

8. The array polarization type three-dimensional live cell high-resolution holographic dynamic microscopy imaging system according to claim 3, characterized in that The objective lens (6) uses a low-magnification objective lens and is responsible for providing imaging resolution and a wide imaging field of view.

9. The array polarization type three-dimensional living cell high-resolution holographic dynamic microscopic imaging system according to claim 1, characterized in that, It further includes a tube lens (8); the tube lens (8) and the first lens (9) form an image-space telecentric optical path and are responsible for broadening the original polarized light beam into a parallel optical path with a wider range.

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