The experimental platform of the differential phase contrast of the present invention based on the colorful multiplexing method can be built based on any commercial microscope system, and only needs to add a light source modulation module color LCD display or programmable color LED in the optical path. Array. Taking the differential phase lining imaging system based on LCD display light as an example, the principle of the microscope system is schematicallyfigure 2 (a) shows, including color multiplexed light source modules (components of mercury lamps, LCDs, condensed mirrors), samples, microscopy (chromoples), bobbin, and color cameras. The color multiplexed light source module can be used in two structures, the first kind of light source using a microscope, the LCD display and the condenser as the lighting module, the act of the LCD is modulated to the light source, so that the light source irradiated on the sample is the present invention Designed color multiplexed pattern. The second is to use the LED as a lighting system, and the color multiplexing light pattern is displayed directly through the computer control LED, and the concentrating mirror is aggregated on the sample. A number of point light sources are included in the LED array or LCD display, and they are ruled to form a two-dimensional matrix. Each point light source can be red (R), green (G), blue (B) three-channel illumination, typical wavelength is 632 nm, green 522 nm and blue light 470 nm. The central spacing D between each point source is typically 1-10 mm. The lighting module is placed under the sample stage, and the upper surface spacing H of the stage is typically between 30-90 mm, and the optical path center is in the optical axis of the microfiber.
If system illumination is performed using the LED array, drive the LED array to light the implementation circuit of each point source, and existing techniques such as single-chip microcomputer, ARM, or programmable logic devices, specific implementation methods can be referred to. Document (Guo Bao Zeng, Deng Yumei: Design of LED Display Control System Based on FPGA [J]. LCD and Display, 2010,25 (3): 424-428). If system illumination is used using an LCD display, use the LCD replacement of the aperture stop under the original microscope, by displaying the illumination pattern of the present invention as a spatial light filter, the technology used in the drive circuit is basically in the LED array. No different, specific implementation methods can be referred to related literature (Lin Fei, Zhang Wen Wen: Rheinburg photo based on programmable LCD, obvious micro-principle and system design. Optical newspaper, 2016, 8: 237-243).
Combinefigure 1 , The present invention is based on a single frame difference method of a single frame difference of color multiplexed illumination, and the steps are as follows:
Step 1, color reuse illumination image acquisition: Using computer via serial control High Contrast LCD display TFT-LCD or high-density programmable color LED array display color multiplexed lighting pattern lighting sample, such asfigure 2 (b) shown. The illumination pattern is a red (R), green (g), blue (b), two colors of red (B), a half-ring illumination of the three colors of the non-symmetric shaft, and the numerical aperture of the semi-annular illumination is equal to the objective intensity, and the lighting intensity is distributed in sinusoidal distribution. . Assuming the numerical aperture of the objective lens is represented as NAObj Then, the color multiplexed lighting function represented in the polar coordinates is:
Sr(ρ, θ) = δ (ρ-NAObj ) SIN (θ + θr)
Sg(ρ, θ) = δ (ρ-NAObj ) SIN (θ + θg)
Sb(ρ, θ) = δ (ρ-NAObj ) SIN (θ + θb)
(r= Θg-120 °, θb= Θg+ 120 °)
Where Sr(ρ, θ), sg(ρ, θ), sb(ρ, θ) respectively indicate the lighting function corresponding to the three wavelengths of red (R), green (G), and blue (B), respectively, indicate the radius and pole angle of the polar coordinate system, respectively, θr, Θg, ΘbThe angle of the asymmetric axis of red (R), Green (G), and blue (B) is respectively. δ (ρ-NAObj ) The shape indicating the illumination pattern is an annular shape of the illumination numerical aperture and the objective numerical aperture phase. As can be seen from this lighting function, as long as the crossword of the three wavelength illumination patterns is 120 °, the design of the present invention is satisfied.
Generate synchronous trigger signals to color cameras while sending lighting control, collecting a color sample image, such asfigure 2 (c), record Ic. The present invention adopts the optimal illumination scheme to achieve a single frame differential phase lining image, which significantly improves the degree of homology of imaging, and greatly enhances its low frequency imaging contrast and high frequency resolution.
If the alternating lighting strategy is used, the lighting pattern will be rotated 90 ° in any direction to collect the second color sample image, remember toc，⊥. The color multiplexing illumination pattern of the single frame is used as the pattern 1, and it rotates from 90 ° in any direction as pattern 2, and the two illumination patterns are used to alternate two images. The phase recovery can be achieved by using the two images to obtain a completely interstitial phase transfer function to achieve completely interstitial imaging resolution.
Step 2, Image Color Channel Separation and Correction: Colorful Sample Images IcSingle-channel separation and color leak correction, resulting in red (R), green (G), blue (B) three channels corresponding to sample strength image Ir, Corr , Ig, corr , IB, CORR ,Such asfigure 2 (D1),figure 2 (d2),figure 2 (D3).
If an alternating lighting strategy is used, the two color samples to collect will be collected.c, Ic，⊥The channel separation and correction is performed, and the two images corresponding to red (R), green (G), blue (B) three-channel sample intensity image Ir, Corr , Ig, corr , IB, CORR , IR, ⊥, corr , Ig, ⊥, CORR , IB, ⊥, CORR.
Since the color LCD or LEDs typically have a wide emit spectrum, and for most color image sensors, the spectral response of different color channels cannot be completely isolated. Thus, light of some color in the lighting may leak into other color channels and detected by the other color channels of the camera, that is, the single-channel image of the color sensor is actually a mixed image of different channels. In color multiplexing, the illumination light of three channels simultaneously illuminate a sample to collect a color image, and since the overlap of the emission spectrum (the partial spectral response of green light is overlapping the blue and red channel), the color leakage Be more obvious. Differential phase lining phase recovery directly after separating channels, color leakage will result in severe phase estimation errors. In order to reduce the phase error due to color leakage, the present invention employs a color leak correction method, indicating the detector signals measured in the color channel as the light of the desired color and the light of the light of the other colors. In other words, the measurement signal in the red (R), Green (G), and the blue (B) channel can be written as:
Ir, Ig, IbIt is the signal strength of the red (R), Green (G), and Blue (B) channel measured by the camera sensor, that is, IcThe intensity image of red (R), green (G), and blue (B) channel is performed directly. Ir, Corr , Ig, corr , IB, CORR It is the light intensity of the red (R), green (G), blue (B) channel incident on the camera sensor, that is, the image intensity of the phase recovery should be subjected to phase recovery. element Indicates the detection response of the LED light of the camera of M (M = R, G, B) color channel to the color of N (n = r, g, b).
The purpose of color leak correction is to get each Numerical, this can be collected according to the cameracGet ir, Corr , Ig, corr , IB, CORR. The specific correction scheme is to illuminate the LEDs of a single color L (L = R, G, B) in the case of not placing the sample, and the color image I collects corresponding to the color camera.c,l', For this image Ic,l'Separation of channels, you can get three channels of images il,r', Il,g', Il,b', Calculate the intensity mean of three channel images, and normalize the mean of the images corresponding to the image of the light color L, which is normalized as the standard, and the three values are obtained. (m = r, g, b). Do this operation for three channels will get all Numerical value. Once the camera is acquired, the channel can be separated to get Ir, Ig, Ib, According to the following formula, the light intensity image I of each wavelength after correction can be obtained.r, Corr , Ig, corr , IB, CORR :
The camera's spectral response matrix can be used to calculate the camera's spectral response matrix, which can be used for subsequent correction images, which effectively alleviate the phase reconstruction error brought by color leaks. For the same set of imaging systems, the spectral response matrix only needs to be calculated.
Step 3, differential phase lining image spectrum generation: II for three channelsr, Corr , Ig, corr , IB, CORR Fourier transform, resulting in the spectral distribution of three images. In order to eliminate the influence of the background item, the spectrum distribution of the differential phase lining image of the sample is obtained, the zero frequency of the spectrum is removed, that is, the zero frequency of the three spectrum is set to 0, the effect of the background item is eliminated, and the differential phase liner of three channel samples is obtained. Image spectrum distribution is expressed as
If an alternating lighting strategy is used, the sample intensity image of a single channel corresponding to two capture images is used.r, Corr , Ig, corr , IB, CORR , IR, ⊥, corr , Ig, ⊥, CORR , IB, ⊥, CORRSolve the spectrum and remove the zero frequency of the spectrum, and the elimination of background items.
Step 4, the phase transfer function calculates: based on weak phase approximation conditions, according to the lighting function and the parameters of the objective lens, calculate the phase transfer function corresponding to different wavelengths. PTFr(ρ, θ), PTFg(ρ, θ), PTFb(ρ, θ).
If an alternating lighting strategy is used, the phase transfer function corresponding to the two lighting patterns is solved PTF.r(ρ, θ), PTFg(ρ, θ), PTFb(ρ, θ), PTFr,⊥(ρ, θ), PTFg,⊥(ρ, θ), PTFb,⊥(ρ, θ).
Such asimage 3 As shown, for any illumination and aperture function, the transmission response of any point Q in the phase transfer function can be obtained by the overlapping region of the solution mirror light pupil function and the off-axis illumination aperture. This is because the lights falling in these areas ensures point q at P (U + Uj) = 1 or Q in P (U-Uj) = 1. However, it is worth noting that when the q point irradiation is close to the central axis of the objective lens, correspond to P (U + Uj) And P (U-UjThe two areas will be offset each other. Therefore, for the location of different Q, the integral partition of the phase transfer function should be divided, such asimage 3 (a),image 3 (b) shown. The solution of phase transfer function is performed as an example of lighting in a single direction, and the solving expression of phase transfer can be obtained:
According to this calculation expression, the lighting function belongs to the calculation of red (R), green (G), blue (B), the transfer function corresponding to the three wavelengths, respectively:
PTFr(ρ, θ) = sin (α)r) SIN (θ + θr)
PTFg(ρ, θ) = sin (α)g) SIN (θ + θg)
PTFb(ρ, θ) = sin (α)b) SIN (θ + θb)
(r= Θg-120 °, θb= Θg+ 120 °)
Α hererΑgΑbNumerical aperture NA from the objective lensObj And illumination wavelength λrΛgΛbDecided, you can solve it according to the following formula:
Step 5. Sample Quantitative Phase Recovery: PTF according to the phase transfer function of different wavelengthsr(ρ, θ), PTFg(ρ, θ), PTFbDifferential phase lining image spectrum of (ρ, θ) and sample The Tikhonov criterion is used to perform reverse volume calculation, resulting in a high resolution spectrum of the sample phase, and the high resolution spectrum is reversed for the transformation, resulting in the quantitative phase distribution of the sample φ:
The K here represents different wavelength channels, which are red (R), green (G), blue (B). PTFk*(ρ, θ) means PTFk(ρ, θ) conjugate distribution. λk/ λ represents the wavelength normalization coefficient, because the phase and wavelength are inversely ratio, so in the method of using color multiplexing, the wavelength is required to normalize the unified phase distribution, and the λ is indicated to normalize the wavelength, which can be selected. For any wavelength, the wavelength of blue illumination is usually selected as a normalized wavelength. β is regularized parameters, generally selects a smaller value, such as 0.01.
If the alternating lighting strategy, the sample image spectrum and transfer function corresponding to the two lighting patterns should be brought into the reverse volume, then the solving formula of the quantitative phase distribution φ of the sample is:
In order to compare the imaging performance of the present invention,Figure 4 The existing single frame differential phase lining imaging is shown, which is compared with the phase transfer function of the present invention, which is uniform, sinusoidal, and sinusoidal ring, which are 120. °. The simulation is used to simulate the multi-axis synthesis phase transfer function corresponding to each illumination pattern, such asFigure 4 (A1),Figure 4 (a2),Figure 4 (A3) shown. Comparing these three phase transfer functions, there is a large amount of missing, and the phase transfer function transmission response should be existed under uniform circular illumination, while the transmission response of its low frequency and high frequency is very weak. This lighting is used to imaging, the phase contrast is poor, the imaging resolution will be lost, and the highest resolution cannot be properly recovered. Compared to uniform circular illumination, the isotropy of the phase transfer function under sinusoidal circular illumination is greatly improved, but the low frequency and high frequency response of its transfer function are still very weak. The sinusoidal lighting of the present invention greatly enhances the respective interesidity of the phase transfer response while the transmission response is significantly enhanced over the entire non-coherent imaging range.Figure 4 (C1),Figure 4 (C2) showsFigure 4 (A3) andFigure 4 (A1), Figure 4 (A3) andFigure 4 The response difference of the phase transfer function of (A2) can clearly observe the lift of the illumination scheme of the present invention for the phase transfer function. In addition, further comparing the phase transfer response of alternating lighting strategy, the result is displayedFigure 4 (b1),Figure 4 (b2),Figure 4 (B3). Comparing these three phase transfer functions, it can be found that the illumination scheme of the present invention can obtain a completely intended phase transfer function at alternating lighting strategies.Figure 4 (D1),Figure 4 (d2) showsFigure 4 (b3) andFigure 4 (b1),Figure 4 (b3) andFigure 4 The response difference of the phase transfer function of (b2), the alternating scheme of the present invention significantly enhances the response of the phase transfer function.
In order to verify the high resolution, high stability, and high contrast of the single-frame differential liner linear quantitative phase imaging result, the standard USAF phase resolution plate is used as a sample, and the uniform circular illumination and the present invention are employed. Methods have been compared experiments, and the experimental results are shownFigure 5 in. Such asFigure 5 (a) andFigure 5 (c) an image collecting a colorful reuse illumination of the present invention.Figure 5 (b1) andFigure 5 (D1) The quantitative phase result obtained under uniform circular illumination, the quantitative phase result of the present invention is shownFigure 5 (b2) andFigure 5 (D2). ComparedFigure 5 (b1) andFigure 5 (b2),Figure 5 (D1) andFigure 5 (D2), it can be found that there is a better robility, better contrast, and a higher contrast, and a higher contrast. Further extraction of the highest resolution phase value plot curve to quantitatively compare the imaging effect of both illumination.Figure 5 (e)Figure 5 (b1) andFigure 5 The highest resolution in (B2) can be found that the reconstruction phase of the present invention can be clearly distinguished in all directions, and the resolution of uniform circular illumination cannot be distinguished.Figure 5 (f)Figure 5 (D1) andFigure 5 The quantitative phase distribution in the highest resolution in (D2) can be found that the present invention has achieved a theoretical maximum resolution of 435 nm, and this resolution cannot be restored under uniform circular illumination.
In order to verify the real-time dynamic imaging effect of the quantitative phase imaging of the present invention based on color multi-use illumination, the cell-cultured cervical cancer (HELA) cells were carried out in vitro, which was placed in a suitable In the culture environment, the reconstructed experimental phase results areFigure 6 Indicated.Figure 6 (a) shows the reconstruction phase results of the full field of view, select two of the two interested zones to zoom in, such asFigure 6 (b) andFigure 6 (c) shown. It can be seen that cells and subcellular information such as nucleus, vesicles can be clearly observed, which will illustrate the high resolution of real-time dynamics. Further display of dynamic cells from different momentsFigure 6 In (d), it can be seen that the phase of the cells at different times is clearly reproduced, and there is no moving artifact or trailing phenomenon.