Digital holographic microscopy of highly sensitive living cells

Abstract

In order to solve the problem that the existing living cell microscopy technology can not display the detailed information of cells, a high sensitivity digital holographic living cell microscopy technology is proposed in this paper. By measuring the phase distribution and refractive index distribution of living cells, the data of living cells are extracted and converted into digital hologram of living cells. Simulation and comparison of the commonly used two-dimensional living cell microscope methods. The experimental results show that the high-sensitivity digital holographic microscopic detection method can obtain the detailed information of living cells, which proves the effectiveness of this study.

Keywords

High sensitivity bioactive cells digital holography microscopy cellular electron microscopy adsorption cell-surface interaction biological refractive index

1. Introduction

Digital holographic microscope (DHM) has the advantages of no fluorescent label, wide field of view, high throughput, and suitable for irregular and various materials. It can realize real-time three-dimensional observation of multiple micro scale samples, and is very suitable for observing the three-dimensional dynamic behavior of micro particles, microorganisms and cells. However, the three-dimensional motion of samples such as nanoparticles, bacteria and cells have different characteristics in different micro application scenarios, so there are different requirements for the three-dimensional positioning of digital holographic microscope, especially the working distance and positioning accuracy of optical axis positioning [1].

In recent years, with the development of high-resolution CCD, high-speed computer and advanced digital image processing technology, high-speed and high-resolution digital processing of hologram has become possible, and digital holography technology has developed rapidly. Its main research areas are: the improvement of experimental technology and reconstruction algorithm; Digital holographic microscope; Phase shift digital holography; Digital holographic encryption technology; Particle flow separation [2]. With the further development of image acquisition experiment, the application of digital holography will enter more fields. Digital holography is a new technology based on the principle of digital holography. In the research of digital holography, scholars at home and abroad mainly study the axial and lateral resolution, reconstruction methods and so on.

In reference [3], digital holographic imaging technology is proposed. Its basic principle is to record hologram by using photoelectric sensor instead of traditional holographic recording material, and then realize holographic reconstruction and digital processing of recorded object by simulating optical diffraction process with computer. But the effect of this method is poor. In reference [4], a digital holographic imaging system using synthetic aperture technology to improve spatial resolution is proposed. In this imaging system, the light source irradiates the object from multiple angles, at the same time, combined with multi-channel restoration technology, the hologram of the light source incident from different angles is collected. Finally, the image resolution of the imaging system is improved according to the synthetic aperture technology. However, the parameters obtained in the study often fail to meet the corresponding accuracy requirements.

In this paper, a high sensitive technique of digital holographic living cell microscope is proposed. The innovation of this method is to use two-step subtraction to correct the phase distortion. The principle of the two-step subtraction method is to record two holograms with or without objects to obtain the phase distribution of the hologram, and then subtract to obtain the phase image without distortion [5]. Not only the clear morphology of static biological samples, but also the morphology and dynamic changes of living cells were observed.

2. Digital holographic microscopy of highly sensitive living cells

2.1 Data extraction of living cells under digital holographic microscope

At present, digital holography micro imaging technology has been able to quantitatively measure the phase distribution and refractive index distribution of transparent samples, such as living cells. It can reach the lateral resolution of diffraction limit and axial accuracy of sub wavelength, but in fact, it can only be called 2.5-dimensional imaging, not the real three-dimensional imaging [6, 7]. The phase image obtained in traditional digital holography is two-dimensional distribution, and the phase value represents the optical path difference, that is, the light passing through the sample, along the beam propagation direction is related to the accumulation of refractive index at each point inside the sample [8, 9, 10]. The obtained refractive index is not along each point of the light, but an average refractive index value. When the refractive index inside the sample and between the sample and the surrounding medium changes very little, under the approximate condition of the refractive index integral along the straight path of the optical path, the plane phase diagram obtained by digital holography meets the following relationship:

$\displaystyle\phi(x,y)=\int{\frac{2\pi}{\lambda}\Delta n(x,y)d}$ (1)

where, $\lambda$ is the incident plane wave length, $\Delta n(x,y)d$ is the spatial distribution of the refractive index difference between the sample and its surrounding medium, $\smallint$ represents the signal transmission frequency. $\pi$ represents the parameter nodes of the digital holographic microscopy method. and z-axis is the optical axis direction.

Therefore, in order to obtain the true three-dimensional distribution of refractive index and accurately give the internal structure of the sample, the internal structure is reconstructed by tomography. The projection of sample refractive index in different directions can be compared with computed tomography (CT). CT technology is to reconstruct the three-dimensional structure of the object according to the tilt image of a series of ray beams in different angles, that is, the projection reconstruction of the three-dimensional structure [11]. The projection is made along the two-dimensional section of the object to all directions in the plane, and then an image of the two-dimensional section is reconstructed by using a certain mathematical model and reconstruction algorithm from this series of projection values. Therefore, it is reasonable and feasible to combine digital holography with CT technology to realize tomographic imaging.

Biological living cell data extraction, as shown in Fig. 1.

Figure 1.

Bioactive cell data extraction principle.

The recording surface of CMOS camera is ( $x$ , $y$ ) plane, and the rotation axis of sample is $y$ axis. At a certain rotation angle position $\theta_{i}$ of the sample, the camera records the interference pattern and reproduces the projection phase diagram $\phi_{i}(x,y_{i},\theta_{i})$ , where $\theta_{i}=\{{0,\Delta\theta,2\Delta\theta,\ldots 180^{\circ}-\Delta\theta}\}$ and $\Delta\theta$ is the rotation angle increment. In the same section, the matrix $\phi(x,y_{i},\theta)$ composed of the same row of phase data extracted from the projection phase group is called the C sinogram of the section [12]. The sinusoid is used as the input data of the algorithm to perform the tomographic reconstruction algorithm to reconstruct the phase distribution side $\phi(x,y_{i})$ of the corresponding section. It can be approximated by solving the minimum norm or the best least square interpolation operator. The commonly used weighting methods in data space help to introduce a priori knowledge to the position of the feature region, so as to generate super-resolution images and then the refractive index distribution $n(x,y_{i})$ of the section can be obtained according to the transformation relationship of Eq. (1).

According to the principle of biological cell data extraction, where ( $x_{0}$ , $y_{0}$ ) is the object plane, ( $x_{0}$ , $y_{0}$ ) is the record plane, and $n$ is the distance from the object to the record plane [13]. The complex amplitude distribution of the light field after the unit amplitude of monochromatic parallel light incident vertically on the measured object is. $O(x,y)$ and $R(x,y)$ are the complex amplitude distribution of object light wave and reference light wave on the recording plane, respectively [14]. The total light field obtained on the recording plane is:

$\displaystyle U(x,y)=\phi(x,y)+R(x,y)$ (2)

In the formula, $R$ representing the cell data measurement parameters. When two beams of light interfere in the recording plane, the intensity distribution can be written as follows:

$\displaystyle H(x,y)=|{\phi(x,y)+R(x,y)}|^{2}$ (3)

In the traditional optical holography, the commonly used recording medium is silver salt photographic film. After exposing the interference patterns of two wavefront, the hologram is obtained by developing. In digital holography, the hologram is directly recorded by digital recording device and stored in computer. At present, the commonly used recording device is CCD or CMOS [15]. In Eq. (3), the first two terms are the intensity distribution of the object light and the reference light, which we call the direct term or the zero level term; The third term is called the conjugate term or the $-$ 1 level term; The fourth term contains the correct complex amplitude information of the object light wave, which we call the $+$ 1 level term.

In digital holography, Fourier transform holograms or Fresnel holograms are usually recorded. At present, the most commonly used recording optical path in off-axis digital holography is lensless Fourier transform optical path and off-axis Fresnel optical path [16].

In the process of recording optical path of lensless Fourier transform digital holography, in this optical path, the reference light is spherical light wave, its point source is located on the object plane, which can be expressed as $\delta({x}_{0}-{x}_{r},y_{0}-{y}_{r})$ , $d$ is the distance from the object to the hologram plane, that is, the recording distance.

In the paraxial approximation, according to the Fresnel diffraction integral formula, the distribution of the object light field on the hologram plane is obtained

$\displaystyle O(x,y)=\int\!\!\!\int_{\infty}\phi_{0}{(x}_{0},y_{0})\exp\left\{% {\frac{jk}{2d}[(x-x_{0})+({y}-{y}_{0})^{2}]}\right\}H(x,y)$ (4)

where $\frac{jk}{2d}$ is Fourier transform.

The spherical reference light wave on the recording plane can be expressed as:

$\displaystyle R(x,y)=\exp\left[{\frac{jk}{2d}(x^{2}+y^{2})}\right]\exp\left[{-% \frac{jk}{d}O(x,y)}\right]$ (5)

The interference between the object light wave and the reference light wave occurs in the recording plane. The intensity distribution $H(x,y)$ of the hologram can be calculated by Eq. (2). Equations (3) and (5) are introduced into Eq. (2). It is found that because the reference point source is coplanar with the object, the secondary phase factor in front of the formula is eliminated [17]. The $+$ 1 term containing the correct object information in lensless Fourier transform hologram can be expressed as:

$\displaystyle OR=\exp\left[{\frac{jk}{d}R(x,y)}\right]$ (6)

In off-axis digital holography, real image, virtual image and zero order image are separated in the spectrum space, which is convenient to extract information, so it is the most commonly used digital holography imaging method [18]. The recording optical path of off-axis Fresnel digital holography is shown in Fig. 2. Digital holography is a technology that uses digital photosensitive electronic devices such as CCD or CMOS cameras to replace the silver salt dry plate in traditional optical holography to record holograms, and uses a computer to simulate the optical holographic reproduction process to accurately reproduce objects. This is a kind of holography surgery. In this optical path, the reference light is an inclined plane light wave:

$\displaystyle R(x,y)=\exp\left[{-j2\pi\left(\frac{\cos\alpha}{\lambda}x+\frac{% \cos\beta}{\lambda}y\right)}\right]$ (7)

where $\alpha$ and $\beta$ are the angle between the reference light and the optical axis. According to the Fresnel diffraction integral formula, the object light wave in the hologram plane can be expressed as:

$\displaystyle O(x,y)=\exp\left[{\frac{jk}{2d}(x^{2}+y^{2})}\right]$ (8)

The light intensity distribution $H(x,y)$ of off-axis Fresnel hologram is obtained by taking Eqs (7) and (8) into Eq. (2). The order term containing the correct information can be expressed as:

$\displaystyle OR=\exp\left[{j2\pi\left(\frac{\cos\alpha}{\lambda}x+\frac{\cos% \beta}{\lambda}y\right)}\right]\exp\left[{\frac{jk}{2d}(x^{2}+y^{2})}\right]% \int\!\!\!\int_{\infty}{O(x_{0},y_{0})}$ (9)

The holographic microscopic data of living cells were obtained.

2.2 Transformation of living cell digital hologram

In order to improve the magnification, in the digital holography, we often add a micro objective lens to the off-axis Fresnel digital holography optical path, and first image the object. This optical path is called pre amplified off-axis Fresnel digital holography recording optical path, as shown in Fig. 2.

Figure 2.

Prevention of large off-axis holographic recording.

$(x_{0},y_{0})$ is the object plane, $(x_{MO},y_{MO})$ is the microscopic object mirror (here we analyze it as a thin lens), and $(\zeta,\eta)$ is the image plane. $(x,y)$ is the recording plane, that is, the hologram plane [19]. $d$ is the distance from the hologram plane to the image plane, that is, the reconstruction distance.

Usually we place the camera on the left or right side of the image plane to record the pre amplified off-axis Fresnel hologram. When we place the camera directly on the image plane, the recorded image is the image plane hologram, which is a special case of pre amplified off-axis Fresnel hologram.

Let the complex amplitude distribution of the object be $O_{0}(x_{0},y_{0})$ , according to the Fresnel diffraction integral formula, through the free space propagation of distance, the object light field distribution on the left side of the micro objective plane is:

$\displaystyle O_{MO}(x_{MO},y_{MO})=\exp\left[{\frac{jk}{2d_{1}}(x_{MO}^{2}+y_% {MO}^{2})}\right]OR$ (10)

If the focal length of the objective lens is ${f}$ , the transmittance function of the objective lens is as follows:

$\displaystyle T(x_{MO},y_{MO})=\exp\left[{-\frac{jk}{2f}(x_{MO}^{2}+y_{MO}^{2}% )}\right]$ (11)

If the focal length of the objective lens is $f$ , $M O$ represents the cell analysis parameters. The transmittance function of the objective lens is as follows:

$\displaystyle O(x,y)=\exp\left[{\frac{jk}{2d_{3}}(x^{2}+y^{2})}\right]T({x}_{% MO},y_{MO})$ (12)

On the recording plane, the slanted parallel reference light can be expressed as:

$\displaystyle R(x,y)=\exp\left[{-j2\pi\left(\frac{\cos\alpha}{\lambda}x+\frac{% \cos\beta}{\lambda}y\right)}\right]$ (13)

For the hologram obtained by interference of object light and reference light, Eqs (12) and (13) are substituted into the formula, and the $+$ 1 level term of pre amplified off-axis Fresnel digital hologram is obtained as follows:

$\displaystyle OR=\exp\left[{j2\pi\left(\frac{\cos\alpha}{\lambda}x+\frac{\cos% \beta}{\lambda}y\right)}\right]R(x,y)$ (14)

The sample coordinates and physical coordinates during the calculation are shown in Fig. 3.

Figure 3.

Schematic diagram of transformation between sample coordinates and physical coordinates.

When the parallel light is focused on the focus through the Fourier lens, the distribution of the object light received on the CCD target surface, namely the back focus surface, is as follows:

$\displaystyle U(x,y)_{1}=\exp\left[{jk\frac{x^{2}+y^{2}}{2f}}\right]\int\!\!\!% \int_{-\infty}{g(x,y)\exp\left[{-2\pi j\left(\frac{x}{\lambda f}+\frac{y}{% \lambda f}\right)}\right]}(u,v)$ (15)

In the above formula, ${u,v}$ is the spatial frequency, $\frac{{x}}{\lambda{f}}$ , $\frac{{y}}{\lambda{f}}$ , and the reference light is the parallel light. In order to separate the reconstructed image and the conjugate image, the angle between the object light and the reference light must be ensured [20, 21, 22]. The field distribution of the reference light on the back focal plane is $R(x,y)=R_{0}\exp[j2\pi by]$ .

After the interference of object light and reference light, the total light field distribution on the back focal plane is as follows:

$\displaystyle U(u,v)=\exp\left[{\frac{x^{2}+y^{2}}{2f}}\right]U(x,y)_{1}$ (16)

The intensity distribution received on the CCD target is:

$\displaystyle I(u,v)=R_{0}^{2}+|G|^{2}+R_{0}G\exp\left[{\frac{{x}^{2}+{y}^{2}}% {2f}}\right]$ (17)

In the above formula, $R_{0}$ represents a clear value of the parallel light. the third term is the spatial spectrum of the original object, and the fourth term is the conjugate spectrum. These two spectrum distributions are transmitted from two columns of plane waves to different directions [23, 24, 25]. The reproduction is performed with the same reproduction light as the original reference light. Since the reference light is a parallel light, the amplitude of the reproduction plane wave can be assumed to be 1 in digital reproduction. So to get the original object, only one inverse Fourier transform is needed for the intensity distribution $I(u,v)$ received by CCD.

$\displaystyle U_{1}(x_{1},y_{1})=I(u,v)[j2\pi(ux+vy)]$ (18)

In the formula, the first term is the 8-function, which represents the bright spot produced by the direct transmission light converging in the center of the image plane through the lens; The second term is the autocorrelation function of the complex amplitude distribution of the object light wave, which forms the halo light near the focus; The third term is the complex amplitude of the original image; The fourth term is the complex amplitude of the conjugate image, and the original image and the conjugate image are symmetrical about the center point. Thus, the digital holography micrograph of living cells with high sensitivity can be obtained.

3. Simulation experiment

3.1 Experimental preparation

In order to verify the effectiveness of the high-sensitivity live-cell digital holography method, a simulation experiment was designed. And compared with the high-sensitivity live-cell digital holography method and the commonly used two-dimensional live-cell digital holography method. In the experiment, a coaxial optical path was used to record multiple phase-shift holograms in each sub-region. After recording each sub-region, control the movement of the sample, record adjacent sub-regions, and adjacent sub-regions overlap. The experiment is developed on the basis of the transmission digital holographic microscopy experiment. The coaxial optical path is adopted, that is, the target light wave is in the same direction as the reference light wave; a phase shift device is introduced in the reference arm, and the phase shift hologram is recorded by controlling the phase shift step length; The measured sample is placed on a two-dimensional electronically controlled translation platform. The translation platform can realize the two-dimensional movement of the sample, and the sample must be photosensitive to the CCD. The sample in the experimental device is placed on a one-dimensional mobile platform. In the experiment of a large-field high-resolution digital holographic microscope, the light source is still a solid-state laser produced by Shanghai GROHE Company, with adjustable wavelength, maximum power and power. The design of the large field of view high-resolution digital full-definition micro-experiment is based on the transmissive digital full-millimeter micro-experiment. Coaxiality is realized by adjusting the incident direction of the target light wave and the reference light wave. Shift, place the camera on the image surface, and record the coaxial phase shift image hologram. Place the sample on the two-dimensional mobile platform. By controlling the movement of the platform, the movement of the sample and the different sub-areas are recorded.

It is a mature high-precision phase-shifting technology to realize step-by-step phase-shifting using piezoelectric ceramics. As an important comparative test data. However, how to effectively compensate the nonlinear, hysteresis, creep and other effects of piezoelectric ceramics, and how to ensure the stability and accuracy of the displacement within the accuracy range of tens of wavelengths, are still the biggest problems in the current phase-shifting technology. To solve this problem, on the one hand, we need to improve the performance of piezoelectric ceramics; On the other hand, it is necessary to improve the performance of control power supply, control circuit and software. We use a series of piezoelectric ceramic models imported from Germany. The specific parameters are shown in Table 1. The controller adopts the digital precision positioning controller produced by Harbin core tomorrow Technology Co., Ltd., adopts modular design, integrates driving power, piezoelectric ceramic micro displacement detection module and control module. The driving module drives the piezoelectric ceramic, and the sensor module detects and processes the feedback signal of the sensor. The core control module precisely controls the experiment, which is used for the precise positioning control of piezoelectric actuator and workbench. At the same time, the closed-loop and open-loop control methods of the controller are also introduced. In the closed-loop control, the precise control is realized by the built-in algorithm. Closed loop control is selected in the experiment.

Table 1
List of symbols and abbreviations

Symbol abbreviation	Paraphrase
CCD	Charge Coupled Devices
$\int$	Signs for Definite Integrals
CT	Computed Tomography
CMOS	Complementary Metal Oxide Semiconductor
exp	Exponential
$\zeta$
$\lambda$	Incident plane wavelength
$\delta({x}_{0}-{x}_{r},y_{0}-y_{r})$	Record distance record distance
$(\zeta,\eta)$	Image plane

The two-dimensional moving device is composed of two-dimensional electronic micro displacement platform and displacement control box. The two-dimensional electronic micro displacement platform is built by two one-dimensional electronic displacement platforms with the same model and phase number. Each one-dimensional displacement platform is a displacement platform produced by Beijing Zhuohanguang company. The imported linear bearing is adopted for the ball screw guide rail of the conversion stage, and the left and right limit switches with protection function and the zero position photoelectric switches are set with the interface. The standard interface motor is equipped with a hand wheel at the back, which can be manually adjusted and the standard mounting hole position is suitable for forming a multi-dimensional electric control displacement table.

Image acquisition adopts a camera produced by Canada company. The experimental control software and data processing software based on ten are compiled. The data processing software is developed to realize the complex amplitude reconstruction, least square unwrapping operation based on cosine transformation, phase compensation and other operations of each phase-shifting sub hologram, and also the splicing operation of sub hologram and sub hologram. As for the processing algorithm of sub hologram, we have introduced in the front section in detail, and the splicing operation algorithm will be described in section. The experimental control software realizes the automatic control of CCD camera, piezoelectric ceramic and two-dimensional translation table, and realizes the linkage control of the three.

3.2 Experimental result

Firstly, the biological teaching sample film of the mosquito mouth apparatus is selected as the imaging object, the recording distance is 12.1 cm, and the hologram is obtained by CCD recording. The hologram is cut into a square, and the size is 2672 pixel $\times$ 2672 pixel, as shown in Fig. 4a.

Then, the numerical reconstruction is carried out in the computer, and the full field amplitude reconstruction image of the hologram obtained directly by one fast Fourier transform is shown in Fig. 4b.

There is a constraint relationship between the pixel size of the reconstructed image and the pixel size of the hologram. $\Delta x^{\prime}=\lambda d/(M\Delta x)$ , $\Delta y^{\prime}=\lambda d/(N\Delta y)$ ( $\Delta x$ and $\Delta y$ are the pixel size of CCD, $M$ and $N$ are the total number of pixels of the hologram). Therefore, the pixel size of the reconstructed image is 2.67 !gym. It should be pointed out here that since the biological samples of the mosquito’s mouth organ are labeled, fixed and not completely transparent, the image of the object can also be obtained from the amplitude image. The original image in the upper right corner is as shown in Fig. 4c.

The corresponding phase distribution of the package is shown in Fig. 4d.

Figure 4.

Cell microscopic effect diagram.

According to Fig. 4, it can be clearly seen that the offset circular fringe corresponds to the secondary phase factor. Then the corresponding hologram, full field amplitude reconstruction image, original image and corresponding wrapped phase image are obtained. It can be seen from Fig. 4d and e that after holographic recording of cells according to the photo above, the cell phase can be obtained by four-step phase-shifting method, and the enlarged cell phase diagram can be obtained clearly. In the solution of cell phase by inverse trigonometric function, the phase is truncated in the main value range of inverse trigonometric function, and the wrapped cell phase is obtained. The actual cell phase can only be obtained after the phase is unwrapped. The least square phase unwrapped method is used. After the phase unwrapped, the remaining spherical wave phase needs to be removed, and the surface fitting method is used to remove it. The effect picture of two-dimensional living cell microscopy and high sensitive living cell digital holography microscopy is shown in Fig. 4e and f.

It can be seen that distortion-free high quality phase images are obtained with high resolution to see clear details of the mosquito mouth. The most elaborate structure is about 24 $\mu$ m. It can be seen that Fig. 4e has more 3-dimensional information than Fig. 4d, which better shows the details of the live cells. By testing the problem of displaying cell details under different methods, the contrast structure is shown in Fig. 5.

Figure 5.

Cell information shows the number data comparison results.

Analysis Fig. 5 shows that the literature [4] method reaches 63, and the literature [5] method is 52, while the reality of the present method is as high as 83. Therefore, the present method is significantly higher than that of the other two methods. And we prove the effectiveness of this method.

4. Discuss

Microscopic digital holography can obtain the phase information of microscopic objects, which can not be obtained by ordinary microscope. Therefore, digital holographic microscopy is of great significance in life science and other fields. Aiming at the key problems of digital holographic microscopic imaging technology, the image quality has been studied, and important achievements have been made in improving the image quality. On this basis, the design of reflection and transmission digital holographic microsystem is completed, and the complete hardware system, software control system and data information processing system of digital holographic microsystem are established, and the digital reconstruction and quantitative analysis of phase images are realized. A phase distortion correction algorithm based on improved mathematical model is proposed for phase contrast microimaging of digital holography, which can automatically eliminate the main phase distortion. The traditional living cell microscope and the high sensitivity living cell digital holographic microscope were compared. The experimental results show that this method can obtain the detailed information of living cells well. The reason is that by measuring the phase distribution and refractive index distribution of living cells, the data of living cells can be extracted and the digital hologram of living cells can be converted. The simulation results were compared with the conventional two-dimensional living cell microscope. The practice proves that this method is effective and has wide application value.

5. Conclusions

The overall design and construction of digital holographic microtomography system include: the structure of projection acquisition light path is analyzed, which provides a basis for selecting sample rotation light path; Based on the optical path of Mach-Zenhder off-axis digital holographic micrograph recording image surface, the sample environment in the optical path of the object is designed and configured, and the approximate Fourier satisfaction is established. In the light path environment of blade projection slice theory, the method of sample location and marking was designed, and the corresponding method of sample displacement error correction was proposed. In the projection acquisition optical path system, an automatic rotation device of sampling Angle based on single chip microcomputer is added. A corresponding interactive control program is developed based on MATLAB language to realize the linkage control of sample rotation and digital holographic acquisition.

Based on the theory of Fourier projection slice, the theoretical model of digital holographic micro tomography is studied. This paper introduces the basic principle and computer implementation steps of reconstructing the internal refractive index distribution of an object by using digital holographic micro projection data and filter back projection reconstruction algorithm. The reconstruction accuracy, computational efficiency and anti noise robustness of the algorithm are studied and optimized. The feasibility and reliability of combining digital holography with tomography to reconstruct the refractive index distribution inside the object are verified by numerical simulation.

This method includes not only primary and secondary items, but also multiple cross items. The algorithm can accurately fit the phase distortion and can be calculated by non iterative algorithm. This method greatly simplifies the calculation process of surface fitting. Then, based on the pre amplified off-axis Fresnel digital holographic optical path, a miniaturized fiber coupled inverted digital holographic microscope system is constructed. Using the phase aberration correction algorithm proposed in this paper, phase contrast imaging of living cell samples grown in culture dish is realized.

In order to solve the problem of incomplete display of cell details by traditional cell microscopy, a high sensitivity method of living cell digital holographic microscope was proposed.

(1)

Traditional microscopic analysis methods of living cells inevitably have the problem of low resolution in the analysis process, which can not display the detailed information of cells more clearly. In order to solve this problem and improve the effect of holographic processing on the number of living cells, a high sensitivity digital holographic microscopic detection method for living cells is proposed in this paper.

(2)

By measuring the phase distribution and refractive index distribution of the transparent living cells, the data of the living cells are extracted and converted into the digital hologram of the living cells. A simulation experiment was designed and compared with the two-dimensional living cell microscope.

(3)

The experimental results show that the proposed method can obtain the detailed information of living cells better than the traditional method, which proves the effectiveness of this study and provides a certain reference value for the biomedical field.

(4)

In the future, it is necessary to further study the problem of detail loss of biological cells in the process of digital holography, so as to maximize the retention of details and improve the applicability of this method in practice.

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Digital holographic microscopy of highly sensitive living cells

Abstract

Keywords

1. Introduction

2. Digital holographic microscopy of highly sensitive living cells

2.1 Data extraction of living cells under digital holographic microscope

3.1 Experimental preparation

Table 1 List of symbols and abbreviations

5. Conclusions

References

Table 1
List of symbols and abbreviations