Microscopic imaging method of phase contrast and differential interference contrast based on the transport of intensity equation

Abstract

A microscopic imaging method of phase contrast (PC) and differential interference contrast (DIC) based on the transport of intensity equation (TIE) includes capturing three intensity images along the optical axis; solving the TIE by deconvolution to obtain the quantitative phase; obtaining the intensity image under the DIC imaging mode according to the DIC imaging principle; and obtaining the corresponding phase image of PC imaging mode according to the PC imaging principle. The method can endow the bright-field microscope with the ability to realize PC and DIC imaging without complex modification of the traditional bright-field microscope. In addition, it has the same imaging performance as the phase contrast microscope and differential interference contrast microscope, which are expensive, complex-structure, and has strict environmental conditions.

Claims

1. A microscopic imaging method of phase contrast (PC) and differential interference contrast (DIC) based on the transport of intensity equation (TIE) comprising the steps of: acquring three intensity images along an optical axis, including a positive defocus image I.sub.z-P, an infocus image I.sub.0, and a negative defocus image I.sub.z-N; obtaining a first-order axial differentiation ∂I/∂z of an intensity I using a numerical difference estimation: $\frac{\partial I}{\partial z} = \frac{I_{z - P} - I_{z - N}}{d z},$ where dz is a distance between the positive defocus image I.sub.z-P and the negative defocus image I.sub.z-N in an axial direction Z; obtaining a quantitative phase image based on the TIE, wherein a quantitative phase ϕ is obtained by substituting the obtained first-order axial differentiation ∂I/∂z into TIE; solving the TIE in a differential interference contrast imaging mode, including using the obtained quantitative phase image to obtain an intensity image DIC(x,y) in the differential interference contrast imaging mode based on a DIC imaging theory; and solving a phase transfer function in a phase contrast imaging mode, and solving the intensity image in the phase contrast imaging mode, including using the obtained quantitative phase image to obtain an intensity image I.sub.PC-A(u) by an annular phase contrast method and an intensity image I.sub.PC-B(u) using a phase contrast of apodization method based on a PC imaging theory.

2. The microscopic imaging method according to claim 1, wherein the step of obtaining the quantitative phase image further comprises: solving the TIE using the obtained first-order axial differentiation ∂I/∂z according to: $- k \frac{\partial I}{\partial z} = \nabla .Math. (I \nabla ϕ); and$ obtaining the quantitative phase ϕ by $ϕ = - k \nabla^{- 2} \nabla .Math. (I^{- 1} \nabla \nabla^{- 2} \frac{\partial I}{\partial z}),$ where k is a wave number, k=2π/λ, λ is a wavelength, ∇ is a gradient operator, .Math. denotes dot product, ∇.sup.−2 is an inverse Laplacian operator.

3. The microscopic imaging method according to claim 1, wherein the step of solving the TIE further comprises: (1) passing a beam of light through a Wollaston prism and dividing the beam of light into two coherent optical fields with Δx, Δy shear; (2) any one of the two coherent optical fields producing another phase shift of π/2, while the other one remaining unchanged; (3) interfering and superimposing the two beams to form an intensity interferogram, thereby obtaining a final differential interference contrast image; and (4) obtaining the intensity image DIC(x,y) in the differential interference contrast imaging mode based on Fourier domain sub-pixel panning by:
U(x,y)=√{square root over (I.sub.0)} exp[iϕ(x,y)]
U.sub.Δ(x,y)=exp(iπ/2)FFT.sup.−1{.Math.(u.sub.x,u.sub.y)exp[i2π(u.sub.xΔx+u.sub.yΔy)]}
DIC(x,y)=|U(x,y)+U.sub.Δ(x,y)|.sup.2, where .Math.(u.sub.x,u.sub.y)=FFT{U(x,y)} is a Fourier transform of U(x,y), (u.sub.x,u.sub.y) is a Fourier-domain coordinate corresponding to a space-domain coordinate (x,y), whose shear Δx,Δy values are adjustable; when the shear Δx,Δy is an integer, it can be solved directly in a space domain; when the shear Δx, Δy is a fractional number, it needs to be converted to a frequency domain; a size of the shear Δx,Δy determines convexity and concavity of the object in the differential interference contrast image; and positive and negative of the shear determines a shadow direction of the object in the differential interference contrast image.

4. The microscopic imaging method according to claim 3, wherein the step of solving the TIE further comprises: generating an object optical field by:
U(x,y)=√{square root over (I.sub.0)} exp[iϕ(x,y)], where I.sub.0 is the focus image obtained in the step of acquiring three intensity images, ϕ(x,y) is the ϕ obtained in the step of obtaining the quantitative phase image, obtaining a light field U.sub.Δ(x,y) after shearing Δx,Δy and phase shifting π/2 by:
U.sub.Δ(x,y)=U(x+Δx,y+Δy)=√{square root over (I/(x+Δx,y+Δy))}exp{i[ϕ(x+Δx,y+Δy)+π/2]}, where Δx is an amount of shear along an x-axis, Δy is an amount of shear along a y-axis, and obtaining the intensity image DIC(x,y) formed by U(x,y) and U.sub.Δ(x,y) by:
DIC(x,y)=|U(x,y)+U.sub.Δ(x,y)|.sup.2=|√{square root over (I.sub.0)} exp[iϕ(x,y)]+√{square root over (I/(x+Δx,y+Δy))}exp{i[ϕ(x+Δx,y+Δy)+π/2]}|.sup.2 where Δx,Δy is integer N, a value of the integer is used to adjust an amount of shear and positive and negative of the integer is used to adjust a direction of shear; DIC(x,y) is the obtained DIC intensity image; wherein: {circle around (1)} for a case of uniform intensity where I.sub.0=1, the DIC imaging theory is based on:
U(x,y)=exp[iϕ(x,y)]
DIC(x,y)=|exp[iϕ(x,y)]+exp{i[ϕ(x+Δx,y+Δy)+π/2]}|.sup.2, {circle around (2)} for weakly phased objects for uneven intensity, the DIC imaging theory is based on:
U(x,y)=√{square root over (I.sub.0)} exp[iϕ(x,y)]=√{square root over (I.sub.0)}[1+iϕ(x,y)]
DIC(x,y)=I.sub.0{1+[ϕ(x,y)−ϕ(x+Δx,y+Δy)]}, {circle around (3)} for weakly phased objects for uniform intensity, thus
U(x,y)=exp[iϕ(x,y)]=1+iϕ(x,y)
DIC(x,y)=1+[ϕ(x,y)−ϕ(x+Δx,y+Δy)], {circle around (4)} for a case of slowly varying intensity √{square root over (I.sub.0)}≈√{square root over (I(x+Δx,y+Δy))}, the DIC imaging theory is based on:
DIC(x,y)=|√{square root over (I(x+Δx,y+Δy))}[exp[iϕ(x,y)]+exp{i[ϕ(x+Δx,y+Δy)+π/2]}]|

5. The microscopic imaging method according to claim 1, wherein in the step of solving the phase transfer function, obtaining the intensity image I.sub.PC-A(u) by the annular phase contrast method based on a phase contrast imaging theory comprises: using an annular illumination, wherein a phase difference plate is annular, and an intensity of the object and an optical field of the object U(x,y) generated by a phase recovery are obtained by
U(x,y)=√{square root over (I.sub.0)} exp[iϕ(x,y)], $I (u) = B δ (u) + A (u) A T F (u) + i Φ (u) P T F (u) B = \int \int L (u_{j}) {.Math. P (u_{j}) .Math.}^{2} d^{2} u_{j} A T F (u) = \frac{- \int \int L (u_{j}) [P^{*} (u_{j}) P (u + u_{j}) + P (u_{j}) P^{*} (u - u_{j})] d^{2} u_{j}}{\int \int L (u_{j}) {.Math. P (u_{j}) .Math.}^{2} d^{2} u_{j}}, P T F (u) = \frac{\int \int L (u_{j}) [P^{*} (u_{j}) P (u + u_{j}) - P (u_{j}) P^{*} (u - u_{j})] d^{2} u_{j}}{\int \int L (u_{j}) {.Math. P (u_{j}) .Math.}^{2} d^{2} u_{j}},$ where I.sub.0 is the focus image obtained in the step of acquiring three intensity images, ϕ(x,y) is the phase image ϕ obtained in the step of obtaining the quantitative phase image, u denotes a frequency coordinate in Fourier space; I(u) is an intensity spectrum distribution; B indicates background items; A(u) is an amplitude spectrum distribution of a sample; ATF(u) denotes an amplitude transfer function; Φ(u) is a phase spectrum distribution of the sample; PTF(u) denotes a phase transfer function; u.sub.j is a corresponding frequency shift vector caused by a single tilted illumination, j indicates different angles of illumination; L(u.sub.j) denotes an intensity distribution of an illumination source in a Fourier domain; P(u) is a pupil function of an objective lens; * denotes a conjugate operation, wherein in a case of annular phase contrast, a light source expression for an annular illumination mode L(u) is $L (u) = {\begin{matrix} 1, & k_{2}^{2} < u_{x}^{2} + u_{y}^{2} < k_{1}^{2} \\ 0, & others \end{matrix},$ wherein the pupil function under the annular phase contrast P.sub.PC-A(u) is $P_{P C A} (u) = {\begin{matrix} 1, & k_{1}^{2} < u_{x}^{2} + u_{y}^{2} < k_{0}^{2} \\ 0.25 \times \exp (i \frac{π}{2}), & k_{2}^{2} < u_{x}^{2} + u_{y}^{2} < k_{1}^{2} \\ 1, & u_{x}^{2} + u_{y}^{2} < k_{2}^{2} \\ 0, & others \end{matrix} k, = N A_{i} / λ (i = 0, 1, 2),$ where (u.sub.x,u.sub.y) is a Fourier-domain coordinate corresponding to a space-domain coordinate (x,y), λ is wavelength, k.sub.0 is a radius of a corresponding objective limit, k.sub.1 is an outer diameter of a phase contrast ring, k2 is an inner diameter of the phase contrast ring; an integration area of a ring illumination corresponds to the phase contrast ring, and an annular phase contrast of ATF.sub.PC-A(u) and PTF.sub.PC-A(u) is expressed as $A T F_{P C - A} (u) = \frac{\begin{matrix} - \int \int L (u_{j}) [⁠ P_{P C - A}^{*} (u_{j}) P_{P C - A} (u + u_{j}) + \\ P_{P C - A} (u_{j}) P_{P C - A}^{*} (u - u_{j})] d^{2} u_{j} \end{matrix}}{\int \int L (u_{j}) {.Math. P_{P C - A} (u_{j}) .Math.}^{2} d^{2} u_{j}}$ $P T F_{P C - A} (u) = \frac{\begin{matrix} \int \int L (u_{j}) [P_{P C - A}^{*} (u_{j}) P_{P C - A} (u + u_{j}) - \\ P_{P C - A} (u_{j}) P_{P C - A}^{*} (u - u_{j})] d^{2} u_{j} \end{matrix}}{\int \int L (u_{j}) {.Math. P_{P C - A} (u_{j}) .Math.}^{2} d^{2} u_{j}},$ wherein when the sample is partially illuminated by spatially coherent light, a resulting annular phase contrast intensity spectrum I.sub.PC-A (u) is a sum of an intensity spectra at multiple illumination angles:
I.sub.PC-A(U)=B.sub.PC-Aδ(u)+A(u)ATF.sub.PC-A(u)+iΦ(u)PTF.sub.PC-A(u)

6. The microscopic imaging method according to claim 5, the intensity image I.sub.PC-B(u) is obtained by an apodization phase contrast method in which a pupil function P.sub.PC-B(u) is expressed by a formula of: $P_{P C - B} (u) = {\begin{matrix} 1, & k_{1}^{' 2} < u_{x}^{2} + u_{y}^{2} < k_{0}^{2} \\ \frac{3}{4 (k_{1}^{'} - k_{1})} u_{x} + [1 - \frac{3}{4 (k_{1}^{'} - k_{1})} k_{1}], & k_{1}^{2} < u_{x}^{2} + u_{y}^{2} < k_{1}^{' 2} \\ 0.25 \times \exp (i \frac{π}{2}), & k_{2}^{2} < u_{x}^{2} + u_{y}^{2} < k_{1}^{2} \\ \frac{- 3}{4 (k_{2} - k_{1}^{'})} u_{x} + [1 + \frac{3}{4 (k_{2} - k_{1}^{'})} k_{2}], & k_{2}^{' 2} < u_{x}^{2} + u_{y}^{2} < k_{2}^{2} \\ 1, & u_{x}^{2} + u_{y}^{2} < k_{2}^{' 2} \\ 0, & others \end{matrix} k_{i} = N A_{i} / λ, i = 0, 1, 2,$ ATF.sub.PC-B(u) and PTF.sub.PC-B(u) of the apodization phase contrast are expressed as $A T F_{P C - B} (u) = \frac{\begin{matrix} \int \int L (u_{j}) [P_{P C - B}^{*} (u_{j}) P_{P C - B} (u + u_{j}) + \\ P_{P C - B} (u_{j}) P_{P C - B}^{*} (u - u_{j})] d^{2} u_{j} \end{matrix}}{\int \int L (u_{j}) {.Math. P_{P C - B} (u_{j}) .Math.}^{2} d^{2} u_{j}} P T F_{P C - B} (u) = \frac{\begin{matrix} \int \int L (u_{j}) [P_{P C - B}^{*} (u_{j}) P_{P C - B} (u + u_{j}) - \\ P_{P C - B} (u_{j}) P_{P C - B}^{*} (u - u_{j})] d^{2} u_{j} \end{matrix}}{\int \int L (u_{j}) {.Math. P_{P C - B} (u_{j}) .Math.}^{2} d^{2} u_{j}},$ where k.sub.0 corresponds to the radius of the objective limit, k.sub.1 is the outer diameter of the phase contrast ring with phase delay, k.sub.2 is the inner diameter of the phase contrast ring with phase delay, k′.sub.1 and k′.sub.2 is overall outer and inner diameter of the phase contrast ring; wherein when a sample is partially illuminated by spatially coherent light, an intensity spectrum of the apodization phase contrast I.sub.PC-B(u) is a sum of an intensity spectra at multiple illumination angles:
I.sub.PC-B(u)=B.sub.PC-Bδ(u)+A(u)ATF.sub.PC-B(u)+iΦ(u)PTF.sub.PC-B(u), thereby obtaining an intensity image in a phase contrast PC imaging mode.

7. The microscopic imaging method according to claim 1, wherein in the step of solving the phase transfer function, the intensity image I.sub.PC-B (u) is obtained by an apodization phase contrast method in which a pupil function P.sub.Pc-B(u) is expressed by a formula of: $P_{P C - B} (u) = {\begin{matrix} 1, & k_{1}^{′2} < u_{x}^{2} + u_{y}^{2} < k_{0}^{2} \\ \frac{3}{4 (k_{1}^{'} - k_{1})} u_{x} + [1 - \frac{3}{4 (k_{1}^{'} - k_{1})} k_{1}], & k_{1}^{2} < u_{x}^{2} + u_{y}^{2} < k_{1}^{' 2} \\ 0.25 \times \exp (i \frac{π}{2}), & k_{2}^{2} < u_{x}^{2} + u_{y}^{2} < k_{1}^{2} \\ \frac{- 3}{4 (k_{2} - k_{2}^{'})} u_{x} + [1 + \frac{3}{4 (k_{2} - k_{2}^{'})} k_{2}], & k_{2}^{' 2} < u_{X}^{2} + u_{y}^{2} < k_{2}^{2} \\ 1, & u_{x}^{2} + u_{y}^{2} < k_{2}^{' 2} \\ 0, & others \end{matrix} k_{i} = N A_{i} / λ, i = 0, 1, 2,$ ATF.sub.PC-B(u) and PTF.sub.PC-B(u) of the apodization phase contrast are expressed as $A T F_{P C - B} (u) = \frac{\begin{matrix} - \int \int L (u_{j}) [P_{P C - B}^{*} (u_{j}) P_{P C - B} (u + u_{j}) + \\ P_{P C - B} (u_{j}) P_{P C - B}^{*} (u - u_{j})] d^{2} u_{j} \end{matrix}}{\int \int L (u_{j}) {.Math. P_{P C - B} (u_{j}) .Math.}^{2} d^{2} u_{j}} P T F_{P C - B} (u) = \frac{\begin{matrix} \int \int L (u_{j}) [P_{P C - B}^{*} (u_{j}) P_{P C - B} (u + u_{j}) - \\ P_{P C - B} (u_{j}) P_{P C - B}^{*} (u - u_{j})] d^{2} u_{j} \end{matrix}}{\int \int L (u_{j}) {.Math. P_{P C - B} (u_{j}) .Math.}^{2} d^{2} u_{j}},$ where k.sub.0 corresponds to a radius of an objective limit, k.sub.1 is an outer diameter of a phase contrast ring with phase delay, k.sub.2 is an inner diameter of the phase contrast ring with phase delay, k′.sub.1 and k′.sub.2 is overall outer and inner diameter of the phase contrast ring; wherein when a sample is partially illuminated by spatially coherent light, an intensity spectrum of the apodization phase contrast I.sub.PC-B(u) is a sum of an intensity spectra at multiple illumination angles:
I.sub.PC-B(u)=B.sub.PC-Bδ(u)+A(u)ATF.sub.PC-B(u)+iΦ(u)PTF.sub.PC-B(u), thereby obtaining an intensity image in a phase contrast PC imaging mode.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of the optical path of the microscopic imaging microscope based on the transport of intensity equation for phase contrast and differential interference contrast.

(2) FIG. 2 is a flowchart of the microscopic imaging method of phase contrast and differential interference contrast based on the transport of intensity equation.

(3) FIG. 3 shows the schematic diagram of different phase contrast rings in the phase contrast method. FIG. 3(a) is the phase contrast ring under annular phase contrast; FIG. 3(b) represents the relationship between the amplitude transmittance (T) and the ring radius (r) of the phase contrast ring half-section under annular phase contrast; where FIG. 3(c) shows the relationship between the phase (P) and the ring radius (r) of the phase contrast ring half-section under annular phase contrast (or apodization phase contrast) (positive phase contrast case); where FIG. 3(d) displays the phase contrast ring under apodization annular phase contrast FIG. 3(e) shows the relationship between the amplitude transmittance (T) and the ring radius (r) of the phase difference ring half-section under apodization phase contrast; FIG. 3(f) represents the relationship between the phase (P) and the ring radius (r) of the phase difference ring half-section under annular phase contrast (or apodization phase contrast) (negative phase contrast case).

(4) FIG. 4 displays the simulated images of the microscopic imaging method of phase contrast and differential interference contrast based on the transport of intensity equation. FIG. 4(a1) shows the phase difference plate under the ring phase contrast; FIG. 4(a2) represents the phase difference plate under the apodization phase contrast; FIG. 4(a3) shows the Amplitude Transfer Function (ATF) of the ring phase contrast; FIG. 4(a4) is the ATF of the apodization phase contrast; FIG. 4(b1) shows the Phase Transfer Function (PTF) of the ring positive phase contrast; FIG. 4(b2) is the PTF of the apodization positive phase contrast; FIG. 4(b3) displays the PTF of the annular negative phase contrast; FIG. 4(b4) shows the PTF of the apodization negative phase contrast; FIG. 4(c1) shows the PTF of the annular phase contrast under annular illumination with the numerical aperture of the objective lens of 0.4; FIG. 4(c2) represents the annular phase contrast PTF of the annular illumination with the numerical aperture of the objective lens of 0.65; FIG. 4(c3) shows the annular phase contrast PTF under annular illumination with a numerical aperture of the objective lens of 0.75; FIG. 4(c4) is the annular phase contrast PTF under annular illumination with a numerical aperture of the objective lens of 1.25; FIG. 4(d1) is the apodization phase contrast PTF under annular illumination with a numerical aperture of the objective lens of 0.4; FIG. 4(d2) represents the apodization phase contrast PTF under annular illumination with the objective lens numerical aperture at 0.65; FIG. 4 (d3) shows the apodization phase contrast PTF under annular illumination at a numerical aperture of the objective lens at 0.75; FIG. 4 (d4) expresses the apodization phase contrast PTF under annular illumination at a numerical aperture of the objective lens at 1.25.

(5) FIG. 5 shows a schematic diagram of the HeLa cell results of the present invention. FIG. 5 (a) displays the quantitative phase image obtained from the transport of intensity equation (TIE); FIG. 5 (b) shows the annular positive phase contrast light intensity image; FIG. 5 (c) expresses the apodization positive phase contrast light intensity image; FIG. 5 (d) shows the differential interference contrast (DIC) light intensity image; FIG. 5 (e) represents the annular negative phase contrast light intensity image; FIG. 5 (f) displays the apodization negative phase contrast light intensity image.

DESCRIPTION OF THE PREFERRED EMBODIMENT

(6) As shown in FIG. 1, the present invention is based on a microscopic imaging system equipped with an adjustable lens module with a standard 4F relay system, the actual hardware platform of which is an inverted Olympus IX71 microscope (it is composed of condenser 1, aperture stop 2, condenser lens 3, objective lens 5, and imaging tube lens 6 in FIG. 1). The microscope was equipped with an Olympus camera 10 (Olympus UC50, resolution 2588 pixels×1960 pixels, 3.4 μm/pixel) for image acquisition and an electronically controlled zoom lens (EL-C-10-30-VISLD, Optotune AG) module 11 for precise zooming. This system is controlled by software via the USB interface and ensures the synchronization of the camera 10 acquisition and the electronically controlled zoom lens module 11 zoom in the direction perpendicular to the focal plane (z-axis). The image stack is acquired by camera 10 after passing through a long working distance planar semi-complex achromatic objective 5 (Olympus, LUCPLFLN 40×, NA 0.6). It can be seen from FIG. 1 that the back focal plane of the objective lens 5 coincides with the front focal plane of the imaging tube lens 6, and the imaging plane of the camera 10 is placed at the back focal plane position of the 4F relay system behind the imaging tube 6 (composed of lenses 8, 9 in FIG. 1). During imaging, the sample 4 to be tested on the stage is adjusted to the position of the front focal plane of the objective lens 5 to form an infinity-corrected imaging system. An electronically controlled zoom lens module 11 is placed on the spectrum surface of the standard 4F relay system. The light is condensed by the condenser 1 and becomes part of the coherent light that illuminates the sample 4 to be tested. The sample 4 to be tested is placed on the stage. The light passes through the sample 4, passes through the imaging tube lens 6, and then converges through the electronically controlled zoom. The lens module 11 is modulated to illuminate the imaging plane 7 of the camera 10, and a series of intensity images can be collected.

(7) With reference to FIG. 2, the present invention utilizes the above-mentioned device to realize the phase contrast (PC) and differential interference contrast (DIC) based on the transport of intensity equation (TIE), including the following four steps:

(8) Step 1, three intensity images are acquired along the optical axis, and the process is as follows: three intensity images are acquired along the optical axis: positive defocus image I.sub.z-P, infocus image I.sub.0, and negative defocus image I.sub.z-N. The first-order axial differentiation ∂I/∂z of the intensity I is obtained by using numerical difference estimation

(9) $\frac{\partial I}{\partial z} = \frac{I_{z - P} - I_{z - N}}{d z}$
where dz is the distance between the positive defocus image I.sub.z-P and the negative defocus image I.sub.z-N in the Z axial direction, and the value is generally 5×10.sup.−6 m.

(10) Step 2, obtain the quantitative phase image ϕ based on the TIE. The procedure is as follows: solve the TIE using the first-order axial differentiation ova, obtained in Step 1

(11) $- k \frac{\partial I}{\partial z} = \nabla .Math. (I \nabla ϕ)$
obtain the quantitative phase image ϕ

(12) $ϕ = - k \nabla^{- 2} \nabla .Math. (I^{- 1} \nabla \nabla^{- 2} \frac{\partial I}{\partial z})$
where k is the wave number, it can be expressed as k=2π/λ (λ is the wavelength). And ∇ is the gradient operator, .Math. denotes the dot product, ∇.sup.−2 is the inverse Laplacian operator.

(13) Step 3, solve TIE in the differential interference contrast (DIC) imaging mode. The specific implementation process is:

(14) (1) Firstly, a beam of light passes through a Wollaston prism and divides into two coherent light fields with Δx, Δy shear;

(15) (2) Any one of them produces another phase shift of π/2, the other one remains unchanged;

(16) (3) Finally, the two beams interfered and superimposed to form an intensity interferogram, that is, the final DIC image obtained. The specific process is as follows: First, the resulting object optical field U(x,y) is:
U(x,y)=√{square root over (I.sub.0)} exp[iϕ(x,y)]
where I.sub.0 is the focus image obtained in Step 1, ϕ(x,y) is the ϕ obtained in Step 2, then the light field U.sub.Δ(x,y) after shearing Δx,Δy and phase shifting π/2 is
U.sub.Δ(x,y)=U(x+Δx,y+Δy)=√{square root over (I/(x+Δx,y+Δy))}exp{i[ϕ(x+Δx,y+Δy)+π/2]}
then the intensity image DIC(x,y) formed by U(x,y) and U.sub.Δ(x,y) is
DIC(x,y)=|U(x,y)+U.sub.Δ(x,y)|.sup.2=|√{square root over (I.sub.0)}exp[iϕ(x,y)]+√{square root over (I/(x+Δx,y+Δy))}exp{i[ϕ(x+Δx,y+Δy)+π/2]}|.sup.2
where Δx,Δy is integer N, its value is used to adjust the amount of shear and the positive and negative is used to adjust the direction of shear; DIC(x,y) is the obtained DIC intensity image. The solution for the special case of DIC imaging is as follows:
{circle around (1)} For the case of uniform intensity there has I.sub.0=1, then
U(x,y)=exp[iϕ(x,y)]
U.sub.Δ(x,y)=U(x+Δx,y+Δy)=exp{i[ϕ(x+Δx,y+Δy)+π/2]}
DIC(x,y)=|U(x,y)+U.sub.Δ(x,y)|.sup.2=|exp[iϕ(x,y)]+exp{i[ϕ(x+Δy,y+Δy)+π/2]}|.sup.2
{circle around (2)} For weakly phased objects for uneven intensity, i.e., ϕ(x,y)□ 1, so
U(x,y)=√{square root over (I.sub.0)}exp[iϕ(x,y)]=√{square root over (I.sub.0)}[1+iϕ(x,y)]
DIC(x,y)=I.sub.0{1+[ϕ(x,y)−ϕ(x+Δx,y+Δy)]}
{circle around (3)} For weakly phased objects for uniform intensity, thus

(17) $U (x, y) = \exp [i ϕ (x, y)] = 1 + i ϕ (x, y) U_{Δ} (x, y) = U (x + Δ x, y + Δ y) = \exp {i [ϕ (x + Δ x, y + Δ y) + π / 2]} = \exp (i π / 2) [1 + i ϕ (x + Δ x, y + Δ y)] = i [1 + i ϕ (x + Δ x, y + Δ y)] = i - ϕ (x + Δ x, y + Δ y) D I C (x, y) = {.Math. U (x, y) + U_{Δ} (x, y) .Math.}^{2} = 2 + 2 [ϕ (x, y) - ϕ (x + Δ x, y + Δ y)] + \underset{\underset{\approx 0}{︸}}{ϕ^{2} (x + Δ x, y + Δ y) + ϕ^{2} (x, y)} ▯1 + [ϕ (x, y) - ϕ (x + Δ x, y + Δ y)]$
In this case, ϕ.sup.2(x+Δx,y+Δy)+ϕ.sup.2(x,y) can be approximated as zero, so the DIC intensity image obtained in this case is
DIC(x,y)=1+[ϕ(x,y)−ϕ(x+Δx,y+Δy)]
{circle around (4)} For the case of slowly varying intensity √{square root over (I.sub.0)}≈√{square root over (I(x+Δx,y+Δy))}, there is
DIC(x,y)=|√{square root over (I(x+Δx,y+Δy))}{exp[iϕ(x,y)]+exp{i[ϕ(x+Δx,y+Δy)+π/2]}}|.sup.2.

(18) (4) In practice, however, it is not only limited to integer shear quantities but also fractional shear quantities can be achieved. Then the solution, in this case, must be performed in the frequency domain:
U(x,y)=√{square root over (I.sub.0)} exp[iϕ(x,y)]
U.sub.Δ(x,y)=exp(iπ/2)FFT.sup.−1{.Math.(u.sub.x,u.sub.y)exp[i2π(u.sub.xΔx+u.sub.yΔy)]}
DIC(x,y)=|U(x,y)+U.sub.Δ(x,y)|.sup.2
The above method is a sub-pixel translation based on the Fourier domain, where .Math.(u.sub.x,u.sub.y)=FFT{U(x,y)} is the Fourier transform of, (u.sub.x,u.sub.y) is the Fourier-domain coordinate corresponding to the space domain coordinate (x,y) (i.e., the frequency-domain coordinate). The value of the shear Δx,Δy is adjustable. When the shear Δx,Δy is integer, it can be solved directly in the space domain, and when the shear Δx,Δy is fractional number, it needs to be converted to the frequency domain. Meanwhile, the magnitude of the shear Δx,Δy determines the convexity of the object in the DIC image, which is often within the range of (1,10); and the positive or negative shear value Δy,Δy determines the shading direction of the object in the DIC image.

(19) This step results in an intensity image in the DIC imaging mode DIC(x,y).

(20) Step 4, solve the phase transfer function in the phase contrast (PC) imaging mode, and solve the intensity image in the PC imaging mode by the annular phase contrast method or the apodization phase contrast method.

(21) The specific implementation process is: using the quantitative phase image obtained in Step 2, based on the phase contrast imaging theory to obtain the intensity image I.sub.PC-A(u) obtained by the annular phase contrast method and the intensity image I.sub.PC-B(u) obtained by the apodization phase contrast method. The first method is the annular phase contrast method: compared with the traditional phase contrast method, the difference is that the illumination is annular (instead of plane waves) and the phase contrast plate in the imaging system is annular (instead of a point in the middle) [see FIG. 3(a), FIG. 3(b), FIG. 3(c), and FIG. 3(f) for the specific forms]. The intensity and phase of the object are obtained by phase recovery and the resulting object optical field U(x,y)
U(x,y)=√{square root over (I.sub.0)} exp[iϕ(x,y)]
Thus,

(22) $I (u) = B δ (u) + A (u) A T F (u) + i Φ (u) P T F (u) B = \int \int L (u_{j}) {.Math. P (u_{j}) .Math.}^{2} d^{2} u_{j} AT F (u) = \frac{- \int \int L (u_{j}) [P^{*} (u_{j}) P (u + u_{j}) + P (u_{j}) P^{*} (u - u_{j})] d^{2} u_{j}}{\int \int L (u_{j}) {.Math. P (u_{j}) .Math.}^{2} d^{2} u_{j}} P T F (u) = \frac{\int \int L (u_{j}) [P^{*} (u_{j}) P (u + u_{j}) - P (u_{j}) P^{*} (u - u_{j})] d^{2} u_{j}}{\int \int L (u_{j}) {.Math. P (u_{j}) .Math.}^{2} d^{2} u_{j}}$
where I.sub.0 is the focus image obtained in Step 1, ϕ(x,y) is the phase image ϕ obtained in Step 2, u denotes the frequency coordinate in Fourier space; I(u) is the intensity spectrum distribution; B indicates background items; A(u) is the amplitude spectrum distribution of the sample; ATF(u) denotes the amplitude transfer function; Φ(u) is the phase spectrum distribution of the sample; PTF(u) denotes the phase transfer function; u.sub.j is the corresponding frequency shift vector caused by a single tilted illumination, j indicates different angles of illumination; L(u.sub.j) denotes the intensity distribution of the illumination source in the Fourier domain; P(u) is the pupil function of the objective lens; * denotes the conjugate operation. The above expressions are general expressions for the background term, amplitude transfer function and phase transfer function under complex illumination and are applicable to all illumination modes and pupil functions; therefore, in the case of annular phase contrast, the light source expression for the annular illumination mode L(u) is

(23) $L (u) = {\begin{matrix} 1, & k_{2}^{2} < u_{x}^{2} + u_{y}^{2} < k_{1}^{2} \\ 0, & others \end{matrix}$
The pupil function under the annular phase contrast P.sub.PC-A(u) is

(24) $P_{PC - A} (u) = {\begin{matrix} 1, & k_{1}^{2} < u_{x}^{2} + u_{y}^{2} < k_{0}^{2} \\ 0.25 \times \exp (i \frac{π}{2}), & k_{2}^{2} < u_{x}^{2} + u_{y}^{2} < k_{1}^{2} \\ 1, & u_{x}^{2} + u_{y}^{2} < k_{2}^{2} \\ 0, & others \end{matrix} k_{i} = N A_{i} / λ (i = 0, 1, 2)$
where (u.sub.x,u.sub.y) is the Fourier-domain coordinate corresponding to the space domain coordinate (x,y), λ is the wavelength, k.sub.0 is the radius of the corresponding objective limit, k.sub.1 is the outer diameter of the phase contrast ring, k2 is the inner diameter of the phase contrast ring. In the set of simulation experiments shown in FIGS. 4(a1), 4(a3), 4(b1), 4(b3), and 4(c1), the NA.sub.1=0.28, NA.sub.2=0.25, NA.sub.0=0.4 are used. The ring illumination integration region corresponds to the phase difference ring (inner diameter is k.sub.2, outer diameter is k.sub.1). Then the annular phase contrast of ATF.sub.PC-A(u) and PTF.sub.PC-A(u) can be expressed as

(25) $A T F_{P C - A} (u) = \frac{\begin{matrix} - \int \int L (u_{j}) [P_{P C - A}^{*} (u_{j}) P_{P C - A} (u + u_{j}) + \\ P_{P C - A} (u_{j}) P_{PC - A}^{*} (u - u_{j})] d^{2} u_{j} \end{matrix}}{\int \int L (u_{j}) {.Math. P_{P C - A} (u_{j}) .Math.}^{2} d^{2} u} P T F_{P C - A} (u) = \frac{\begin{matrix} \int \int L (u_{j}) [P_{PC - A}^{*} (u_{j}) P_{P C - A} (u + u_{j}) - \\ P_{PC - A} (u_{j}) P_{PC - A}^{*} (u - u_{j})] d^{2} u \end{matrix}}{\int \int L (u_{j}) {.Math. P_{P C - A} (u_{j}) .Math.}^{2} d^{2} u_{j}}$
The corresponding with ATF.sub.PC-A(u) and PTF.sub.PC-A(u) results are shown in FIG. 4(a3) and FIG. 4(b1). Under complex partially coherent illumination like annular phase contrast, the intensity distribution of the acquired image can be regarded as a linear superposition of the intensity at different angles, so we decompose the complex illumination into illumination at different angles for analysis. At this point, the annular illumination can be represented by the Fourier translation technique, so the Fourier transform of the complex amplitude on the camera plane satisfies
W.sub.j(u)=√{square root over (L(u.sub.j))}[δ(u−u.sub.j)−A(u−u.sub.j)+iΦ(u−u.sub.j)]P(u)
Thus, the spectral distribution of the intensity images acquired by the camera under single-angle tilt illumination can be written as a convolution of W.sub.j(u) and its complex conjugate w.sub.j*(u):
(u)=W.sub.j(u).Math.W.sub.j*(u)=L(u.sub.j)δ(u)|P(u.sub.j)|.sup.2−L(u.sub.j)A(u)[P*(u.sub.j)P(u+u.sub.j)+P(u.sub.j)P*(u−u.sub.j)]+iL(u.sub.j)Φ(u)[P*(u.sub.j)P(u+u.sub.j)−P(u.sub.j)P*(u−u.sub.j)]
In order to linearize the phase recovery, the cross-convolution term in the above equation is neglected. When the sample is partially illuminated by spatially coherent light, the resulting annular phase contrast intensity spectrum I.sub.PC-A(u) can be written as the sum of the intensity spectrum at multiple illumination angles
I.sub.PC-A(u)=B.sub.PC-Aδ(u)+A(u)ATF.sub.PC-A(u)±iΦ(u)PTF.sub.PC-A(u)
For the phase difference plate, the smaller the size, the higher the contrast of the low-frequency component; for the attenuation rate, it can be used to control the ratio of the low-frequency component to the high-frequency component—usually the larger the value taken the greater the attenuation and the higher the phase contrast (90%); for the phase shift amount, there are two cases of positive and negative phase contrast, the difference lies in the phase in the phase difference plate that the delay of the former is +π/2 and the delay of the latter is −π/2. The algorithm can obtain different annular phase contrast images by changing the value of the numerical aperture of the objective NA.sub.0, where the simulation results of changing the numerical aperture of the objective NA.sub.0 are shown in FIG. 4(c1) to FIG. 4(c4).

(26) Another method based on phase contrast imaging theory is the apodization phase contrast method: the difference compared to the annular phase contrast method is the use of an apodization phase contrast plate. By using selective amplitude filters in the phase contrast plate located in the objective lens in the back focal plane adjacent to the phase film, the effect of reducing halos and improving the contrast of the sample can be achieved. These amplitude filters consist of neutral density filter films, and the films are applied to the phase contrast plate around the phase film. The transmittance of the phase shift ring in the latest variant phase difference plate is about 25%, while the pair of adjacent rings around the phase shift ring in the variant phase difference plate has neutral density, and the transmittance varies uniformly from 25% to 100% on both sides [see FIG. 3(d), FIG. 3(e), FIG. 3(c), and FIG. 3(f) for the specific form], and the width of the phase film is the same in both plates. The solution method is similar to the circular phase contrast, the difference is that the former is a step filter and the latter is a trapezoidal filter, i.e., the pupil function P.sub.PC-B(u) is different. It is expressed by the equation

(27) $P_{P C - B} (u) = {\begin{matrix} 1, & k_{1}^{' 2} < u_{x}^{2} + u_{y}^{2} < k_{0}^{2} \\ \frac{3}{4 (k_{1}^{'} - k_{1})} u_{x} + [1 - \frac{3}{4 (k_{1}^{'} - k_{1})} k_{1}], & k_{1}^{2} < u_{x}^{2} + u_{y}^{2} < k_{1}^{′2} \\ 0.25 \times \exp (i \frac{π}{2}), & k_{2}^{2} < u_{x}^{2} + u_{y}^{2} < k_{1}^{2} \\ \frac{- 3}{4 (k_{2} - k_{2}^{'})} u_{x} + [1 + \frac{3}{4 (k_{2} - k_{2}^{'})} k_{2}], & k_{2}^{' 2} < u_{x}^{2} + u_{y}^{2} < k_{2}^{2} \\ 1, & u_{x}^{2} + u_{y}^{2} < k_{2}^{' 2} \\ 0, & others \end{matrix} k_{i} = N A_{i} / λ (i = 0, 1, 2)$
Thus, ATF.sub.PC-B(u) and PTF.sub.PC-B(u) of the apodization phase contrast can be expressed as

(28) 0 $A T F_{P C - B} (u) = \frac{\begin{matrix} - \int \int L (u_{j}) [P_{P C - B}^{*} (u_{j}) P_{P C - B} (u + u_{j}) + \\ P_{P C - B} (u_{j}) P_{P C - B}^{*} (u - u_{j})] d^{2} u_{j} \end{matrix}}{\int \int L (u_{j}) {.Math. P_{P C - B} (u_{j}) .Math.}^{2} d^{2} u} P T F_{P C - B} (u) = \frac{\begin{matrix} \int \int L (u_{j}) [P_{P C - B}^{*} (u_{j}) P_{P C - B} (u + u_{j}) - \\ P_{P C - B} (u_{j}) P_{P C - B}^{*} (u - u_{j})] d^{2} u_{j} \end{matrix}}{\int \int L (u_{j}) {.Math. P_{P C - B} (u_{j}) .Math.}^{2} d^{2} u_{j}}$
where k.sub.0 corresponds to the radius of the objective limit, k.sub.1 is the outer diameter of the phase contrast ring with phase delay, k.sub.2 is the inner diameter of the phase contrast ring with phase delay. In the simulated experimental plots shown in FIG. 4(a2), FIG. 4(a4), FIG. 4(b2), FIG. 4(b4) and FIG. 4(d1), the NA.sub.1=0.28, NA.sub.2=0.25, NA.sub.0=0.4 are used. The phase difference ring with phase delay corresponds to the solid-filled ring in FIG. 3(d), and there is a π/2 phase delay with intensity attenuation effect, and the attenuation form is shown in FIG. 3(e). k′.sub.1 and k′.sub.2 is the overall outer and inner diameter of the phase contrast ring corresponding to the slash-filled ring in FIG. 3(d), there is only an intensity attenuation effect without phase delay, and the attenuation form is shown in FIG. 3(e). The algorithm can be used to adjust the width of the diagonal filling ring in FIG. 3(d), and to obtain different images of the variant phase contrast by changing the value of the numerical aperture of the objective NA.sub.0, where the simulation results of changing the numerical aperture of the objective NA.sub.0 can be seen in FIG. 4(d1) to FIG. 4(d4). The corresponding results of ATF.sub.PC-B(u) and PTF.sub.PC-B(u) are shown in FIG. 4(a4) and FIG. 4(b2). Thus, when the sample is partially illuminated by spatially coherent light, the obtained intensity spectrum of the apodization phase contrast I.sub.PC-B(u) can be written as the sum of the intensity spectrum at multiple illumination angles
I.sub.PC-B(u)=B.sub.PC-Bδ(u)+A(u)ATF.sub.PC-B(u)+iΦ(u)PTF.sub.PC-B(u)

(29) This procedure results in the intensity image in the phase contrast (PC) imaging mode: the intensity image I.sub.PC-A(u) obtained by the annular phase contrast method and the intensity image I.sub.PC-B(u) obtained by the apodization phase contrast method, respectively.

(30) FIG. 5 shows the experimental results of the HeLa cell, i.e., the phase contrast and differential interference contrast intensity images obtained from the quantitative phase image obtained from the TIE shown in FIG. 5(a), using the above algorithm. Among them, FIG. 5(b) displays the annular positive phase contrast intensity map; FIG. 5(c) shows the apodization positive phase contrast intensity map; FIG. 5(d) shows the DIC intensity image; FIG. 5(e) shows the annular negative phase contrast intensity image; and FIG. 5(f) apodization negative phase contrast intensity image. Comparing the phase contrast and differential interference contrast image with the quantitative phase image, it can be seen that the phase contrast and differential interference contrast image obtained by the present algorithm can achieve higher contrast and richer details; comparing the phase contrast and differential interference contrast images via the proposed method with the phase contrast and differential interference contrast images obtained by the traditional method, it can be seen that the present algorithm can achieve the same imaging effect as the traditional phase contrast and differential interference contrast images. In summary, the present invention can endow the bright-field microscope with the ability to achieve phase contrast and differential interference contrast imaging without the need for complex modification of the traditional bright-field microscope. That is, only need to use an ordinary traditional bright-field microscope without adding any complicated components. Through the PC and DIC algorithms, this method has the advantages of quantitative, high-speed, low-cost, simple structure, and less external interference. In addition, it has the same imaging performance as the phase contrast microscope and differential interference contrast microscope, which are expensive, complex-structure, and has strict environmental conditions.

Microscopic imaging method of phase contrast and differential interference contrast based on the transport of intensity equation

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Cpc classification

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G02B21/365

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G02B27/126

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G02B21/367

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H04N23/56

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G02B21/14

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G02B27/12

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