A Biological Imaging Device and Method Based on Wide-Spectrum Defocus Phase Retrieval

Abstract

The present invention provides a biological imaging device and method based on wide-spectrum defocused phase retrieval, so as to solve the technical problems in the prior art that the scanning process is complex and the use of a coherent light source easily introduces interference fringes in the detected light intensity, affecting the imaging clarity of biological samples. The device includes an illumination unit, a beam splitter, a spectral measurement unit and a biological imaging unit. The biological imaging unit includes a fourth lens and a detector sequentially arranged along the transmission optical path of the beam splitter. A biological sample to be measured is placed between the beam splitter and the fourth lens. In this method, the detector receives the corresponding detected light intensity on the negative defocus plane A, the focal plane B and the positive defocus plane C of the fourth lens, and then combines with the phase retrieval method based on wide spectrum to realize the imaging of biological samples. The scanning process of the present invention is simple, uses wide-spectrum illumination, avoids the influence of interference fringes on the imaging clarity during coherent light illumination, and can also realize non-staining and quantitative phase imaging of biological samples.

Description

Technical Field

[0001] The present invention belongs to the field of optics, and relates to a biological imaging device and method, in particular to a device and method for imaging biological samples such as biological sections, cells or bacteria based on wide-spectrum defocus technology and wide-spectrum phase retrieval method. Background Art

[0002] Biological imaging is the most intuitive measurement means to understand the biological structure and function, and is becoming more and more important in the field of clinical medical diagnosis. Whether in the field of biological or medical research, clear and intuitive images are of great significance in exploring the pathogenesis, clinical manifestations and disease diagnosis of diseases.

[0003] Optical microscopy imaging technology can directly observe biological samples and can be roughly divided into three categories:

[0004] One is traditional optical microscopy imaging technology, including bright-field microscopy imaging technology, dark-field microscopy imaging technology and fluorescence microscopy imaging technology. The imaging contrast of bright-field microscopy imaging technology is poor, and staining is required when observing weakly absorbing and weakly scattering biological samples, resulting in loss of biological cell activity; compared with bright-field microscopy imaging technology, the imaging contrast of dark-field microscopy imaging technology is improved, but the imaging light intensity is too weak and the imaging quality is not high; fluorescence microscopy imaging technology can only observe cell structures labeled with fluorescent dyes, and short-wavelength excitation light is likely to cause irreversible phototoxic damage to living cells during the detection of living cells.

[0005] Two is qualitative microscopy imaging technology, including phase-contrast microscopy imaging technology and differential interference contrast microscopy imaging technology. Phase-contrast microscopy imaging technology is mainly used to observe unstained transparent biological samples, converting invisible phase changes into visible intensity changes. Its observation effect is far better than that of bright-field microscopy imaging technology. Its disadvantages are that the halo phenomenon affects cell observation, and the annular beam illumination reduces the utilization rate of the light source. In addition, it is difficult to achieve the modulation of the intensity ratio of transmitted light and diffracted light; while differential interference contrast microscopy imaging technology overcomes the halo phenomenon of phase-contrast microscopy imaging technology and has higher imaging quality, but the optical path system is more complex.

[0006] The third is quantitative microscopy technology. The first two types of microscopy technology can only make qualitative observations, while quantitative microscopy technology can realize quantitative phase measurement, including microscopy technology based on interference, microscopy technology based on intensity transfer equation, and microscopy technology based on iteration. Among them, the microscopy technology based on interference has a complex optical system and has high requirements on the stability of experimental conditions, the coherence of the light source, and the resolution of the interference fringes. In addition, the solution of the phase requires unwrapping, so it has not been widely used; the microscopy technology based on the intensity transfer equation is a non-interference technology. When the light intensity and the axial differential of the light intensity on the plane to be measured are known, the phase information is directly obtained by numerically solving the intensity transfer equation. However, when this technology relies on the phase contrast information within a small defocus range to restore the phase, the phase contrast of the low-frequency part of the phase is low, and using a large defocus will make The phase contrast of the high-frequency part of the phase is weakened; based on iterative microscopy technology, iterative calculations are performed multiple times between the object surface to be measured and the diffraction surface, and amplitude replacement and phase-preserving projection operations are performed until the phase of the object surface to be measured is solved. The representative of this technology is the stacked microscopy technology. Although it has high resolution, the calculation is time-consuming, and the scanning process of the experimental operation is very complicated. In addition, the iterative microscopy technology is a non-interference technology, and the coherent light source used is very likely to introduce interference fringes in the actual detection light intensity, affecting the phase measurement accuracy and ultimately affecting the imaging clarity of the biological sample. Summary of the Invention

[0007] The purpose of the present invention is to solve the technical problems of the existing iterative microscopic imaging technology based on stacked microscopy, such as time-consuming calculations, complex scanning process, and the use of coherent light sources, which easily introduce interference fringes in the actual detection light intensity, affecting the imaging clarity of biological samples. The present invention provides a biological imaging device and method based on wide-spectrum defocused phase recovery.

[0008] In order to solve the above technical problems, the technical solutions provided by the present invention are:

[0009] A bio-imaging device based on wide spectrum defocus phase recovery is special in that it includes an illumination unit, a beam splitter, a spectrum measurement unit and a bio-imaging unit;

[0010] The illumination unit emits a wide spectrum of illumination light which is incident on the beam splitter and is divided into a reflected light beam and a transmitted light beam;

[0011] The spectrum measurement unit includes a third lens and a spectrometer arranged in sequence along the reflection light path of the beam splitter;

[0012] The biological imaging unit comprises a fourth lens and a detector arranged in sequence along the transmission light path of the beam splitter; a biological sample to be tested is placed between the beam splitter and the fourth lens;

[0013] The detector can be moved along the optical axis at the positions of the negative defocus plane A, the focal plane B and the positive defocus plane C of the fourth lens to receive corresponding detection light intensities;

[0014] The transmission direction of the transmitted light beam is defined as the positive direction, the distance between the negative defocus plane A and the focal plane B is -Δz, and the distance between the positive defocus plane C and the focal plane B is Δz. Where f is the focal length of the fourth lens.

[0015] Furthermore, a biological imaging device based on wide-spectrum defocus phase recovery also includes a one-dimensional translation stage, which is used to place a detector. Through the axial translation of the one-dimensional translation stage, the detector can be moved and detected at three positions: the negative defocus plane A, the focal plane B, and the positive defocus plane C of the fourth lens.

[0016] Furthermore, a biological imaging device based on wide spectrum defocused phase recovery also includes a two-dimensional translation stage, which is used to place the biological sample. By moving the two-dimensional translation stage, two-dimensional scanning measurement of the biological sample is achieved to select a suitable area of the biological sample to be tested.

[0017] Furthermore, the lighting unit adopts Köhler illumination, including a white light source, a first lens, a second lens and a filter arranged in sequence along the output light path of the white light source. The white light emitted by the white light source is converged by the first lens, collimated by the second lens, and then filtered by the filter to form a wide-spectrum illumination light. The wide-spectrum illumination light avoids the influence of additional interference fringes on imaging clarity during coherent light illumination.

[0018] Furthermore, the lighting unit further includes a first aperture and a second aperture sequentially arranged between the first lens and the second lens, and the first aperture and the second aperture are used to remove stray light.

[0019] At the same time, the present invention also provides a biological imaging method based on wide spectrum defocused phase recovery, which is special in that it includes the following steps:

[0020] 1) Spectrometer measures the spectral composition of broad spectrum illumination light

[0021] The spectrometer measures the spectral components of the broad spectrum illumination light and obtains the band composition in the broad spectrum illumination light: λ=[λ1,…λ i ,…λ n ],i∈[1,n],λ i Represents the i-th wavelength information, and the weight of each wavelength is α=[α1,…α i ,…α n ], and α1+…α i +…α n =1;

[0022] 2) Detecting light intensity using wide - spectrum defocusing technology

[0023] The detector detects at three positions: the negative defocus plane A at a distance - Δz from the focal plane, the focal plane B, and the positive defocus plane C at a distance Δz from the focal plane, and obtains three corresponding detected light intensities I -Δz (x1,y1), I f (x2,y2) and I Δz (x3,y3); where, (x1,y1), (x2,y2) and (x3,y3) are the coordinates of the negative defocus plane A, the focal plane B, and the positive defocus plane C respectively, and the defocus amount f is the focal length of the fourth lens;

[0024] 3) Achieving biological sample imaging based on wide - spectrum phase retrieval method

[0025] 3.1) The initial assumed light field distribution on the focal plane B is:

[0026] U f (x2,y2) = [α1U f (x2,y2,λ1),…α i U f (x2,y2,λ i ),…α n U f (x2,y2,λ n )], i ∈ [1,n];

[0027] where, U f (x2,y2,λ i ) = exp[jφ f (x2,y2,λ i )], φ f (x2,y2,λ i ) is the initial assumed value of the phase corresponding to the wavelength λ i Define the iteration number as m, m is an integer greater than or equal to 1, and the initial iteration number m = 1;

[0028] 3.2) The light field U f (x2,y2) on the focal plane B is diffracted and transmitted forward to the positive defocus plane C, and the light field distribution on the positive defocus plane C is:

[0029] U Δz (x3,y3) = P weight [U f (x2,y2),Δz] = {α11P[U f (x2,y2,λ1),Δz],…α i P[U f (x2,y2,λ i ),Δz],…α nP[U f (x2, y2, λ n )], Δz};

[0030] Among them, P weight is the multi - wavelength weighted angular spectrum transmission operator, and P is the single - wavelength angular spectrum transmission operator;

[0031] 3.3) Use the actual detected light intensity I Δz (x3, y3) of the positive defocus plane C to replace the calculated light intensity of the positive defocus plane C, retain the phase, and obtain the updated light field distribution U' Δz (x3, y3);

[0032] 3.4) The updated light field U' Δz (x3, y3) of the positive defocus plane C is transmitted backward to the focal plane, and the updated light field distribution of the focal plane B is

[0033] 3.5) Use the actual detected light intensity I f (x2, y2) of the focal plane B to replace the calculated light intensity of the focal plane B, retain the phase, and obtain the updated light field distribution of the focal plane B

[0034] 3.6) The updated light field distribution of the focal plane B is transmitted backward to the negative defocus plane A, and the light field distribution of the negative defocus plane A is

[0035] 3.7) Use the actual detected light intensity I -Δz (x1, y1) of the negative defocus plane A to replace the calculated light intensity of the negative defocus plane A, retain the phase, and obtain the updated light field distribution U' -Δz (x1, y1);

[0036] 3.8) The updated light field U' -Δz (x1, y1) of the negative defocus plane A is transmitted forward to the focal plane B, and the updated light field distribution of the focal plane B is

[0037] 3.9) Use the actual detected light intensity I f (x2, y2) of the focal plane B to replace the calculated light intensity of the focal plane B, retain the phase, and obtain the updated light field distribution of the focal plane B

[0038] 3.10) The updated light field of the focal plane B is transmitted backward to the biological sample plane, and the obtained light field distribution of the biological sample plane is:

[0039]

[0040] Among them, Γ weight is the multi-wavelength weighted lens transmission operator, Γ is the single-wavelength lens transmission operator, (x0, y0) is the coordinate of the biological sample surface;

[0041] 3.11) Calculate the error evaluation function MSE or root mean square RMS. When the error evaluation function MSE or root mean square RMS is less than the corresponding threshold, the iteration stops and executes 3.12); otherwise, m=m+1, Return to 3.2);

[0042] 3.12) According to the updated focal plane B light field Obtain the phase distribution of the biological sample: Realize biological sample imaging, where Angle represents the phase operation.

[0043] Furthermore, in step 3.3), the updated positive defocus plane C light field distribution U' Δz (x3,y3) is obtained by the following formula:

[0044]

[0045] In step 3.5), the updated focal plane B light field distribution Obtained by the following formula:

[0046]

[0047] In step 3.7), the updated light field distribution U' of the negative defocus plane A -Δz (x1,y1) is obtained by the following formula:

[0048]

[0049] In step 3.9), the updated focal plane B light field distribution Obtained by the following formula:

[0050]

[0051] Furthermore, in step 3.11), the calculation formula of the error evaluation function MSE is:

[0052] When m=1, MSE>10 -5 ;

[0053] When m>1,

[0054] The threshold of the error evaluation function MSE is 10 -5 .

[0055] Further, in step 1), the illumination unit adopts Köhler illumination to provide broadband spectral illumination light.

[0056] The beneficial effects of the present invention compared with the prior art are as follows:

[0057] 1. A biological imaging device based on broadband spectral defocus phase retrieval provided by the present invention, in the biological imaging optical path, uses a detector to detect at three positions of the negative defocus plane A, the focal plane B, and the positive defocus plane C of the fourth lens respectively, obtains the corresponding detected light intensities, and combines with the phase retrieval method based on broadband spectrum to solve the phase information of the biological sample, thereby realizing the imaging of the biological sample. Compared with the ptychographic microscopy technology, it does not include a complex scanning process and the experimental operation is simple.

[0058] 2. A biological imaging device based on broadband spectral defocus phase retrieval provided by the present invention adopts broadband spectral Köhler illumination. When compared with coherent light illumination, it will not introduce additional interference fringes to affect the phase measurement accuracy, and improves the imaging accuracy of the biological sample.

[0059] 3. A biological imaging device based on broadband spectral defocus phase retrieval provided by the present invention, the detector is arranged on a one-dimensional translation stage. Through the axial translation of the one-dimensional translation stage, the detector can be conveniently moved to detect at three positions of the negative defocus plane A, the focal plane B, and the positive defocus plane C of the fourth lens, and obtain the corresponding detected light intensities.

[0060] 4. A biological imaging device based on broadband spectral defocus phase retrieval provided by the present invention, the biological sample is arranged on a two-dimensional translation stage. By moving the two-dimensional translation stage, two-dimensional scanning measurement of the biological sample can be realized, which is convenient for selecting a suitable area of the biological sample to be measured.

[0061] 5. A biological imaging method based on broadband spectral defocus phase retrieval provided by the present invention can realize non-staining and quantitative phase imaging of biological samples.

[0062] 6. A biological imaging method based on broadband spectral defocus phase retrieval provided by the present invention has a fast imaging speed and has application prospects in the field of real-time imaging of living biological samples. Description of the Drawings

[0063] Figure 1 It is a schematic structural diagram of an embodiment of the biological imaging device based on broadband spectral defocus phase retrieval of the present invention;

[0064] Figure 2 It is a flowchart of step 3) of an embodiment of the biological imaging method based on broadband spectral defocus phase retrieval of the present invention.

[0065] The description of the reference numerals is as follows:

[0066] 1 - White light source; 2 - First lens; 3 - First aperture; 4 - Second aperture; 5 - Second lens; 6 - Filter; 7 - Beam splitter; 8 - Third lens; 9 - Spectrometer; 10 - Biological sample; 11 - Two-dimensional translation stage; 12 - Fourth lens; 13 - Detector; 14 - One-dimensional translation stage. Detailed implementation manners

[0067] To make the advantages and features of the present invention clearer, the following further elaborates on the present invention in detail with reference to the accompanying drawings and specific embodiments.

[0068] As Figure 1 shown, a biological imaging device based on wide-spectrum defocused phase retrieval of the present invention includes an illumination unit, a beam splitter 7, a spectral measurement unit, and a biological imaging unit; the illumination unit adopts Köhler illumination and includes a white light source 1 and a first lens 2, a first aperture 3, a second aperture 4, a second lens 5, and a filter 6 arranged in sequence along the light-emitting optical path of the white light source 1. The white light emitted by the white light source 1 is converged by the first lens 2 and collimated by the second lens 5, and then filtered by the filter 6 to form wide-spectrum illumination light, providing uniform wide-spectrum illumination light for the biological imaging optical path, avoiding the influence of additional interference fringes on the imaging clarity during coherent light illumination. The first aperture 3 and the second aperture 4 are used to remove stray light; the wide-spectrum illumination light emitted by the illumination unit is incident on the beam splitter 7 and is divided into a reflected light beam and a transmitted light beam; the spectral measurement unit includes a third lens 8 and a spectrometer 9 arranged in sequence along the reflected optical path of the beam splitter 7; the biological imaging unit includes a fourth lens 12 and a detector 13 arranged in sequence along the transmitted optical path of the beam splitter 7; a biological sample 10 to be measured is placed between the beam splitter 7 and the fourth lens 12. Defining the transmission direction of the transmitted light beam as the positive direction, assuming that the focal plane B of the fourth lens 12 is the origin, the distance between the negative defocused plane A and the focal plane B is -Δz, and the distance between the positive defocused plane C and the focal plane B is Δz, where f is the focal length of the fourth lens 12. The detector 13 is arranged on the one-dimensional translation stage 14, and the detector 13 receives the corresponding detected light intensities at three positions of the negative defocused plane A, the focal plane B, and the positive defocused plane C of the fourth lens 12 respectively by axially translating the one-dimensional translation stage 14; the biological sample 10 is arranged on the two-dimensional translation stage 11, and by moving the two-dimensional translation stage 11, two-dimensional scanning measurement of the biological sample 10 is realized to select a suitable measurement area of the biological sample 10.

[0069] After the white light emitted by the white light source 1 of the illumination unit is converged by the first lens 2 and collimated by the second lens 5, it forms broadband illumination light after passing through the filter 6. The beam splitter 7 splits the broadband illumination light into two paths: a reflected beam and a transmitted beam. The reflected beam enters the spectrometer 9 through the third lens 8 to form a spectral measurement optical path, and the spectrometer 9 measures the spectral components of the broadband illumination light. The biological imaging unit realizes the two-dimensional scanning measurement of the biological sample 10 by moving the two-dimensional translation stage 11 to select a suitable measurement area of the biological sample 10. The transmitted beam after being split by the beam splitter 7 irradiates the biological sample 10 to be measured. After being converged by the fourth lens 12, the detector 13 axially translates through the one-dimensional translation stage 14 and detects at three positions: the negative defocus plane A, the focal plane B, and the positive defocus plane C of the fourth lens respectively, and receives the corresponding detected light intensities. Using the multiple detected light intensities detected by the detector 13 and the phase retrieval method based on broadband spectrum, the imaging of the biological sample can be realized.

[0070] The present invention also provides a biological imaging method based on broadband spectrum defocus phase retrieval, including the following steps:

[0071] 1) The spectrometer 9 measures the spectral components of the broadband illumination light

[0072] The illumination unit adopts Köhler illumination. The broadband illumination light emitted is reflected by the beam splitter 7. The reflected beam enters the spectrometer 9 through the third lens. After being measured by the spectrometer 9, the wavelength bands in the broadband illumination light are as follows: λ = [λ1,····λ i ,…λ n , i ∈ [1, n], λi i represents the i-th wavelength information, and the weights of each wavelength α = [α1,…α i ,…α n , and α1 + …α i + …α n = 1;

[0073] 2) Obtaining the detected light intensity by the broadband spectrum defocus technique

[0074] By moving the two-dimensional translation stage 11, the two-dimensional scanning measurement of the biological sample 10 is realized. First, a suitable measurement area of the biological sample 10 is selected; the transmission direction of the transmitted beam is defined as the positive direction. The transmitted beam of the broadband illumination light emitted by the illumination unit after being split by the beam splitter 7 irradiates the biological sample 10 to be measured. After being converged by the fourth lens 12, the detector 13 axially translates through the one-dimensional translation stage 14 and sequentially detects at three positions: the negative defocus plane A at a distance of -Δz from the focal plane, the focal plane B, and the positive defocus plane C at a distance of Δz from the focal plane, and obtains the corresponding three detected light intensities I -Δz (x1, y1), I f (x2, y2) and I Δz(x3, y3); where, (x1, y1), (x2, y2), and (x3, y3) are the coordinates of the negative defocus plane A, the focal plane B, and the positive defocus plane C respectively, and the defocus amount where f is the focal length of the fourth lens (12);

[0075] 3) Implementing biological sample imaging based on the wide-spectrum phase retrieval method

[0076] The specific steps for implementing biological sample imaging based on the wide-spectrum phase retrieval method are as follows, and the corresponding algorithm flow is as Figure 2 shown:

[0077] 3.1) According to the band composition and weights in the wide-spectrum illumination light obtained in step 1), the initial assumed expression for the light field distribution on the focal plane B is:

[0078] U f (x2, y2) = [α1U f (x2, y2, λ1), … α i U f (x2, y2, λ i ), … α n U f (x2, y2, λ n )], i ∈ [1, n];

[0079] where U f (x2, y2, λ i ) = exp[jφ f (x2, y2, λ i )]; where φ f (x2, y2, λ i ) is the initial assumed value of the phase corresponding to the wavelength λ i . Define the number of iterations as m, m is an integer greater than or equal to 1, and the initial number of iterations m = 1;

[0080] 3.2) The light field U f (x2, y2) on the focal plane B is diffracted and transmitted forward to the positive defocus plane C, and the light field on the positive defocus plane C is U Δz (x3, y3) = P weight [U f (x2, y2), Δz] = {α1P[U f (x2, y2, λ1), Δz], … α i P[U f (x2, y2, λ i ), Δz], … α n P[U f (x2, y2, λ n )], Δz}; where, P weightis the multi - wavelength weighted angular spectrum transmission operator, representing that the optical fields corresponding to each wavelength are diffracted and transmitted to the positive defocus plane C respectively. P is the single - wavelength angular spectrum transmission operator; U is obtained by calculating according to the weight Δz (x3, y3);

[0081] 3.3) Use the actual detected optical intensity I Δz (x3, y3) received by the detector 13 to replace the calculated optical intensity of the positive defocus plane C, retain the phase, and obtain the updated optical field distribution of the positive defocus plane C:

[0082] 3.4) The updated optical field U' Δz (x3, y3) of the positive defocus plane C is transmitted backward to the focal plane B, and the updated optical field distribution of the focal plane B is

[0083] 3.5) Use the actual detected optical intensity I f (x2, y2) received by the detector 13 to replace the calculated optical intensity of the focal plane B, retain the phase, and obtain the updated optical field distribution of the focal plane B:

[0084] 3.6) The updated optical field distribution of the focal plane B is transmitted backward to the negative defocus plane A, and the optical field distribution of the negative defocus plane A is

[0085] 3.7) Use the actual detected optical intensity I -Δz (x1, y1) received by the detector 13 to replace the calculated optical intensity of the negative defocus plane A, retain the phase, and obtain the updated optical field distribution of the negative defocus plane A:

[0086] 3.8) The updated optical field U' -Δz (x1, y1) of the negative defocus plane A is transmitted forward to the focal plane B, and the updated optical field distribution of the focal plane B is

[0087] 3.9) Use the actual detected optical intensity I f (x2, y2) received by the detector 13 again to replace the calculated optical intensity of the focal plane B, retain the phase, and obtain the updated optical field distribution of the focal plane B as:

[0088] 3.10) The updated optical field of the focal plane B is transmitted backward to the biological sample plane, and the optical field distribution obtained on the biological sample plane is: where, Γ weight$\Gamma$ is the multi-wavelength weighted lens transmission operator, $\gamma$ is the single-wavelength lens transmission operator, and $(x_0, y_0)$ are the coordinates of the biological sample surface;

[0089] 3.11) Calculate the error evaluation function MSE or the root mean square RMS, and determine whether to stop the iteration according to whether the error evaluation function MSE or RMS is less than the corresponding threshold;

[0090] In this embodiment, the error evaluation function MSE is calculated, and the error evaluation function MSE is calculated according to the following formula:

[0091] When $m = 1$, MSE > 10 -5 ;

[0092] When $m>1$,

[0093] When the error evaluation function MSE < 10 -5 the iteration stops and step 3.12) is executed; otherwise $m = m + 1$, return to 3.2);

[0094] 3.12) Obtain the phase distribution of the biological sample 10 based on the updated light field in the focal plane B : Implement the imaging of the biological sample 10, where Angle represents the phase calculation operation.

[0095] The above is only used to illustrate the technical solution of the present invention and is not intended to limit it. For those of ordinary skill in the art, the specific technical solution recorded in the above embodiments can be modified, or some technical features can be equivalently replaced. These modifications or replacements do not cause the essence of the corresponding technical solution to deviate from the scope of the technical solution protected by the present invention.

Claims

1. A biological imaging method based on wide spectrum defocus phase recovery, characterized in that: The following steps are involved: 1) Build a bioimaging device and measure the spectral composition of broad-spectrum illumination light The biological imaging device comprises an illumination unit, a beam splitter (7), a spectrum measurement unit and a biological imaging unit; the illumination unit emits a wide spectrum of illumination light incident on the beam splitter (7) and is divided into a reflected light beam and a transmitted light beam; the spectrum measurement unit comprises a third lens (8) and a spectrometer (9) arranged in sequence along the reflected light path of the beam splitter (7); the biological imaging unit comprises a fourth lens (12) and a detector (13) arranged in sequence along the transmitted light path of the beam splitter (7); a biological sample (10) to be tested is placed between the beam splitter (7) and the fourth lens (12); the detector (13) receives corresponding detection light intensities at the positions of the negative defocus plane A, the focal plane B and the positive defocus plane C of the fourth lens (12); the transmission direction of the transmitted light beam is defined as the positive direction, the distance between the negative defocus plane A and the focal plane B is -Δz, the distance between the positive defocus plane C and the focal plane B is Δz, Wherein f is the focal length of the fourth lens (12); The spectrometer (9) measures the spectral components of the wide-spectrum illumination light and obtains the wavelength composition of the wide-spectrum illumination light: λ=[λ1,····λ i ,…λ n ],i∈[1,n],λ i Represents the i-th wavelength information, and the weight of each wavelength is α=[α1,····α i ,…α n ], and α1+…α i +…α n =1; 2) Wide spectrum defocusing technology to obtain detection light intensity The detector (13) detects in sequence at three positions: a negative defocus plane A at a distance of -Δz from the focal plane, a focal plane B, and a positive defocus plane C at a distance of Δz from the focal plane, and obtains three corresponding detection light intensities I -Δz (x1,y1),I f (x2,y2) and I Δz (x3, y3); where (x1, y1), (x2, y2) and (x3, y3) are the coordinates of the negative defocus plane A, focal plane B and positive defocus plane C respectively; 3) Imaging of biological samples based on wide-spectrum phase retrieval method 3.1) Initially assume that the light field distribution at focal plane B is: U f (x2,y2)=[α1U f (x2,y2,λ1),…a i U f (x2,y2,λ i ),…a n U f (x2,y2,λ n )], i∈[1,n]; Among them, U f (x2,y2,λ i )=exp[jφ f (x2,y2,λ i )],φ f (x2,y2,λ i ) is the wavelength λ i The corresponding phase assumes an initial value, and the number of iterations is defined as m, where m is an integer greater than or equal to 1, and the initial number of iterations m=1; 3.2) Light field U at focal plane B f The forward diffraction of (x2, y2) is transmitted to the positive defocus plane C, and the light field distribution of the positive defocus plane C is: U Δz (x3,y3)=P weight [U f (x2,y2),Δz]={α1P[U f (x2,y2,λ1),Δz],…α i P[U f (x2,y2,λ i ),Δz],…a n P[U f (x2,y2,λ n )],Δz}; Among them, P weight is the multi-wavelength weighted angular spectrum transmission operator, and P is the single-wavelength angular spectrum transmission operator; 3.3) Actual detection light intensity I using positive defocus plane C Δz (x3, y3), replace the calculated light intensity of the positive defocus plane C, retain the phase, and obtain the updated positive defocus plane C light field distribution U' Δz (x3,y3); 3.4) Updated positive defocus plane C light field U' Δz (x3, y3) is transmitted back to the focal plane, and the updated light field distribution of focal plane B is 3.5) Actual detection light intensity I at focal plane B f (x2, y2), replace the calculated light intensity of focal plane B, retain the phase, and obtain the updated light field distribution of focal plane B 3.6) Updated light field distribution at focal plane B The light field distribution of the negative defocus surface A is transmitted in reverse direction. 3.7) Actual detection light intensity I using negative defocus plane A -Δz (x1, y1), replace the calculated light intensity of the negative defocus surface A, retain the phase, and obtain the updated light field distribution U' of the negative defocus surface A -Δz (x1,y1); 3.8) Updated negative defocus plane A light field U' -Δz (x1, y1) is forwarded to the focal plane B, and the updated light field distribution of the focal plane B is 3.9) Actual detection light intensity I at focal plane B f (x2, y2), replace the calculated light intensity of focal plane B, retain the phase, and obtain the updated light field distribution of focal plane B 3.10) Updated focal plane B light field The light field distribution on the biological sample surface is obtained by reverse transmission: Among them, Γ weight is the multi-wavelength weighted lens transmission operator, Γ is the single-wavelength lens transmission operator, (x0, y0) is the coordinate of the biological sample surface; 3.11) Calculate the error evaluation function MSE or root mean square RMS. When the error evaluation function MSE or root mean square RMS is less than the corresponding threshold, the iteration stops and executes 3.12); otherwise, m=m+1, Return to 3.2); 3.12) According to the updated focal plane B light field The phase distribution of the biological sample (10) is obtained: Realize biological sample imaging, where Angle represents the phase operation.

2. The biological imaging method based on wide spectrum offset focus phase recovery according to claim 1, characterized in that: In step 3.3), the updated positive defocus plane C light field distribution U' Δz (x3,y3) is obtained by the following formula: In step 3.5), the updated focal plane B light field distribution Obtained by the following formula: In step 3.7), the updated light field distribution U' of the negative defocus plane A -Δz (x1,y1) is obtained by the following formula: In step 3.9), the updated focal plane B light field distribution Obtained by the following formula:

3. The biological imaging method based on wide spectrum offset focus phase recovery according to claim 2, characterized in that: In step 3.11), the calculation formula of the error evaluation function MSE is: When m=1, MSE>10 -5 ; When m>1, The threshold of the error evaluation function MSE is 10 -5 .

4. The biological imaging method based on wide spectrum offset focus phase recovery according to claim 3, characterized in that: In step 1), the lighting unit adopts Köhler lighting, comprising a white light source (1), a first lens (2), a second lens (5), and a filter (6) arranged in sequence along the outgoing light path of the white light source (1).

5. The biological imaging method based on wide spectrum offset focus phase recovery according to claim 1, characterized in that: In step 1), the biological imaging device further comprises a one-dimensional translation stage (14), and the one-dimensional translation stage (14) is used to place the detector (13).

6. The biological imaging method based on wide spectrum offset focus phase recovery according to claim 5, characterized in that: In step 1), the biological imaging device further comprises a two-dimensional translation stage (11), and the two-dimensional translation stage (11) is used to place the biological sample (10).

7. The biological imaging method based on wide spectrum offset focus phase recovery according to claim 4, characterized in that: In step 1), the lighting unit further comprises a first diaphragm (3) and a second diaphragm (4) which are sequentially arranged between the first lens (2) and the second lens (5).

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