CN104199182B - Two-step diffraction phase imaging method and corresponding phase retrieval method - Google Patents

Abstract

The invention discloses a two-step diffraction phase imaging method and a corresponding phase recovery method, which utilizes the 0th order of grating diffraction and the +1st order and -1 order diffracted light to form a two-step co-geometric optical path interference pattern, which has a high degree of stability, Then calculate the difference between the two interference patterns to eliminate the background light intensity, and then use the Hilbert transform to recover the phase information of the sample. Compared with the phase recovery method commonly used in off-axis interference, the present invention does not require high-pass filtering, high-frequency information is preserved intact, and the phase recovery rate is fast, and the imaging method is applicable to all off-axis interference, including slight off-axis interference. The invention has wide practical value and application prospect in phase microscopy, especially in the application fields of transparent samples such as biological cell phase imaging and phase measurement.

Description

A Two-step Diffraction Phase Imaging Method and Corresponding Phase Restoration Method

technical field

The invention belongs to the technical field of interference microscopic imaging, and in particular relates to a two-step diffraction phase microscopic imaging method based on grating diffraction and a corresponding phase recovery operation method.

Background technique

Optical microscopy technology has opened a door for the observation of microscopic things, and has played an important role in the fields of biology and medicine. Numerous biological samples, such as living cells, are mostly transparent and appear as phase objects. These samples can be imaged clearly and without damage using phase imaging techniques that convert between phase and intensity. At present, interferometric phase microscopy is the mainstream technology, which has the advantages of fast measurement speed and high resolution.

In 2004, Professor Gabriel Popescu of the United States proposed Fourier phase microscopy technology, which uses the scattered light and non-scattered light of the sample as the object field and the reference field respectively, so that coaxial interference of co-geometric optical paths occurs, and combined with phase shifting technology, multiple images are collected. Interferograms for phase imaging. Similar technology also has US patent technology US2009290156 (A1) (Spatial Light Interference Microscope and Cell Tissue Fourier Transform Light Scattering Transmission Method). The phase imaging of this type of technology is highly stable, and the phase recovery operation is simple. The disadvantage is that it is difficult to precisely control the phase shift in practice, and multiple phase shifts are not conducive to the dynamic real-time measurement of the sample. In contrast, off-axis interferometry has a single-shot property, which can be well used for the study of fast phenomena in phase objects. For example, MIT patent technology CN20110374950.7 (system and method for Hilbert phase imaging), it is based on a typical Mach-Zehnder interference optical path, and uses Hilbert integral transformation to process a single interferogram to achieve Interferometric phase imaging. Another example is the digital holographic microscope (DHM-1000) first launched by Lyncee Tec SA in Switzerland in 2006 based on off-axis interference, which can directly observe the three-dimensional shape and phase distribution of samples. However, in this type of technology, the object light and the reference light use separate optical paths to interfere, which is easily affected by external vibration and environmental interference. In this regard, off-axis interferometric phase imaging with a common optical path is proposed. For example, the diffraction phase microscopy technique and its extended techniques utilize the diffraction characteristics of gratings to achieve fast imaging without sacrificing stability. However, like other off-axis interferences, it cannot make full use of the resolution and spatial bandwidth of the CCD, and it needs to eliminate the background image through high-pass filtering during the phase recovery process, which may easily cause the loss of high-frequency information. Then, a kind of slight off-axis interference technology combined with phase shift technology was proposed, such as the two-step slight off-axis interference achieved by Professor Adam Wax of Duke University in the United States by controlling two polarizers. In the phase recovery operation, the technology uses the subtraction of two interference patterns to eliminate unnecessary background images, without the need for high-pass filtering to ensure the integrity of sample information. However, the phase shift needs to be determined by fitting the background fringes afterwards, which increases the complexity of phase recovery to a certain extent. In addition, this technology uses the interference microscope with two optical paths, which does not have the advantage of a common optical path.

To sum up, in the prior art, the phase information can be demodulated from a single off-axis interferogram, but off-axis interferometry cannot make full use of the spatial bandwidth of the CCD, and the commonly used background image high-pass filter will also cause the loss of high-frequency information; although there are some The technology uses two interferograms, such as the two-step slight off-axis interference proposed by Adam Wax mentioned in the article, but it uses a dual-optical path interferometric imaging system, which is unstable and requires devices to achieve phase shift and needs to be measured separately.

Contents of the invention

The purpose of the present invention is to provide a two-step diffraction phase imaging method and a corresponding phase recovery method, to make full use of the CCD spatial bandwidth and avoid background image high-pass filtering, so that it is suitable for both off-axis interference and slight off-axis interference, and enhances the phase imaging Stability, precision and efficiency.

In order to solve the above technical problems, the present invention utilizes the two-step diffraction phase imaging and phase recovery technology of grating diffraction characteristics, and the specific technical scheme is as follows:

A two-step diffraction phase imaging method is characterized in that comprising the following steps:

In the first step, the laser beam passes through the microscopic system to form an enlarged microscopic image, and then passes through the grating (12) located on the imaging screen IP, thereby being divided into many levels of diffracted light containing microscopic image information;

In the second step, the diffracted light is subjected to Fourier transform after passing through the third lens (13), and in the Fourier plane, the first baffle plate (16) is used to block the -1 order diffracted light in the spatial light modulator (14) The filter window only allows the +1 order diffracted light to pass through completely, the 0 order diffracted light is low-pass filtered, and then the +1 order and 0 order diffracted light are carried out Fourier inverse transform through the fourth lens (15), completing the +1 order and 0 order diffracted light. The 0th-order diffracted light is completely filtered, and they are respectively used as sample light and reference light to form the first interference pattern on the CCD (18);

In the third step, on the Fourier plane, the first baffle (16) is removed, and the second baffle (17) is used to block the +1 order diffracted light filter window in the spatial light modulator (14), allowing only -1 order and the 0th-order diffracted light pass through, and then pass through the fourth lens (15), forming a second interference pattern on the CCD (18).

A corresponding phase recovery method of a two-step diffraction phase imaging method, characterized in that it comprises the following steps:

The first step is to record two interferograms

The interference pattern formed by the 0th order and +1st order diffracted light of the grating is

Among them, I _S , I _R are the intensity of the object light wave and the reference light wave respectively, and the sum of the two I ₀ = I _S + I _R is the background light intensity, is the spatially varying phase associated with the sample, and k is the spatial frequency of the carrier fringe. The interference pattern formed by the 0-order and -1-order diffracted light diffracted by the grating is

The second step is to calculate the difference between the two interference patterns to eliminate the background light intensity

The third step is to obtain the complex analysis signal related to the phase

First, carry out the Hilbert transform on formula (3)

Among them, HT is Hilbert transform;

Then the complex analysis signal is

Z＝HT(I ₊₁ -I _-1 )+j[-(I ₊₁ -I _-1 )] (5)

Among them, j is the imaginary unit;

The fourth step is to solve the sample wrapping phase

Among them, Im and Re represent the imaginary part and the real part of the complex number respectively;

The fifth step is to obtain the real continuous sample phase by unwrapping the formula (6).

Use the difference between the two interferograms to eliminate unwanted background light intensity, without high-pass filtering, to ensure the integrity of the sample information, so it is suitable for off-axis and slight off-axis interference.

The working principle of the present invention is as follows: the light beam emitted by the laser transmits forward along the vertical direction through the polarizer to ensure that the light beam is linearly polarized light, and then transmits through the first lens, the pinhole spatial filter and the second lens. Beam expansion and collimation system, the beam after beam expansion and collimation enters the microscopic system composed of sample, adjustable stage, microscopic objective lens and mirror, thus forming an enlarged microscopic image; exiting through the microscopic system The light beam has been converted into a parallel beam, and then continues to be transmitted to the relay mirror along the horizontal direction for correction processing, and then the beam is controlled by the aperture, and the microscopic image is copied to the imaging screen IP. At the same time, a grating is placed on the imaging screen IP, which can obtain multi-level diffraction that contains all the spatial information of the image, and then the diffracted light is separated by a standard 4f spatial filter system composed of the third lens, spatial light modulator and fourth lens +1 order and 0 order diffracted light or -1 order and 0 order diffracted light. Specifically: first, the first baffle is used on the Fourier plane FP of the third lens to block the filter window of the -1st order diffracted light in the spatial light modulator, only allowing the +1st order diffracted light to pass through, and the 0th order diffracted light to be low-pass filtered . According to the characteristics of spatial filtering, the fine structure and sudden changes of the image are mainly caused by high-frequency components, so the 0-order diffracted light through low-pass filtering loses the high-frequency information of the image, then the 0-order diffracted light after the filtering system Order and +1 order diffracted light can be used as reference light and sample light respectively, and then form an interference pattern on the CCD. Secondly, the first baffle is removed, and the second baffle is used to block the filter window of the +1 order diffracted light in the spatial light modulator, allowing all the -1 order diffracted light to pass through, and the 0th order diffracted light to be low-pass filtered, and the separated two The beam forms a second interference pattern on the CCD. Then the two interference patterns are subtracted to eliminate the unnecessary background light intensity, the Hilbert transform is applied to the result to obtain the complex analysis signal related to the sample, and finally the phase of the sample is demodulated to complete the phase imaging.

The invention has beneficial effects. 1. The present invention adopts a diffraction-like phase microscopic imaging optical path, which has the characteristics of a common optical path, is highly stable, and the experiment can be repeated; 2. The present invention uses a suitable spatial light modulator to allow grating diffraction +1-order and 0-order light to pass through and Form the first interference fringe, then allow the grating diffraction-1st order and 0th order light to pass through and form the second interference fringe without phase shift, thus avoiding the difficulty of precise phase shift regulation; 3. The present invention selects different diffraction The grating can realize switching between completely off-axis and slight off-axis interference using the CCD space bandwidth, because the grating constant determines the inclination angle of the two interfering light waves; 4. The present invention uses the subtraction of two interference patterns to eliminate the background image without requiring interference The background image, real image and conjugate image in the pattern are completely separated on the frequency spectrum, and the phase recovery method can be applied to traditional off-axis and slight off-axis interference; 5. In the whole phase recovery process of the present invention, the algebraic operation is firstly used to eliminate the background Compared with the common high-pass filtering method, more information can be obtained, and then a Hilbert transform is used to obtain the complex analysis signal related to the sample. The calculation speed is fast, which is beneficial to the real-time phase information of the sample. Extraction and the study of fast phenomena. Therefore, the present invention has a wide range of applications and has good practical value.

Description of drawings

Fig. 1 is a schematic diagram of the optical path corresponding to the two-step diffraction phase imaging method of the present invention.

In the figure: 1: laser; 2: polarizer; 3: first lens; 4: pinhole spatial filter; 5: second lens; 6: sample; 7: adjustable stage; 8: objective lens; 9: Mirror; 10: relay mirror; 11: aperture; 12: grating; 13: third lens; 14: spatial light modulator; 15: fourth lens; 16: first baffle; 17: second baffle; 18: CCD; IP: Imaging plane; FP: Fourier plane; +1: +1 order diffraction light filter window; -1: -1 order diffraction light filter window; 0 order: 0 order diffraction light filter window; solid black Lines are +1 order diffracted rays; dotted lines are 0th order diffracted rays; double dotted lines are -1 order diffracted rays.

detailed description

Referring to Fig. 1, the two-step diffraction phase imaging method of the present invention is realized by utilizing the diffraction characteristics of the grating and the spatial filtering system.

The beam emitted by the laser 1 is converted into completely linearly polarized light by the polarizer 2, and then enters the light receiving surface of the beam expander collimation system composed of the first lens 3, the pinhole spatial filter 4 and the second lens 5, and passes through The outgoing beam after the beam expander and collimator system is transmitted forward to the sample 6 and the adjustable stage 7, thus becoming the beam carrying the sample information, and then enlarged by the microscopic objective lens 8 and transmitted forward to the mirror 9 to turn into parallel beams. The sample 6, the adjustable stage 7, the microscope objective lens 8 and the reflection mirror 9 can be regarded as an inverted microscope system. The outgoing light beam passing through the microscope system transmits forward sequentially along the horizontal direction through the relay lens 10 for correction processing and beam control through the aperture 11, and then continues to transmit forward to the imaging screen IP, then the microscopic image magnified by the microscope system has been Copy it to the imaging screen IP. At the same time, the grating 12 is placed on the imaging screen IP, which can be divided into many levels of diffracted light containing microscopic image information through grating diffraction, and then the diffracted light continues to transmit forward along the horizontal direction through the third lens 13 and the spatial light modulator 14. A standard 4f spatial filtering system composed of the fourth lens 15. Wherein the spatial light modulator 14 is located on the Fourier plane of the third lens, and has three filter windows: +1 order, 0 order and -1 order diffracted light filter windows for separating +1 order and 0 order The diffracted light is the -1 order and 0 order diffracted light. The specific operation is as follows: first, the first baffle plate 16 is used to block the -1st order filter window in the spatial light modulator 14, and the outgoing diffracted light beam passing through the third lens 13 is forwardly transmitted to the spatial light modulator 14, and the +1st order diffracted light is completely Through the low-pass filtering of the 0th-order diffracted light, the two light beams are used as the object light and the reference light respectively, and then continue to pass through the fourth lens 15 to form the first interference pattern on the CCD18. Secondly, the first baffle 16 is removed, and the second baffle 17 is used to block the +1-order filter window in the spatial light modulator 14, and the -1-order and 0-order diffracted light are selected as object light and reference light respectively, and are used in the CCD18 A second interference pattern is formed on it. This completes the acquisition of two interferograms.

The geometric parameter of the grating 12 grating constant determines the diffraction angle of the grating +1 order and -1 order diffracted light, thus determines the carrier frequency of the interference pattern, and then determines the interference recording method: completely off-axis and slightly off-axis axis.

A phase recovery method corresponding to a two-step diffraction phase imaging method of the present invention is specifically implemented as follows:

The two-step interference patterns formed by the grating diffracting +1st order and 0th order diffracted light and -1st order and 0th order diffracted light can be expressed as:

Among them, I _S and I _R are the intensity of the object light wave (+1 order or -1 order diffracted light) and reference light wave (0 order diffracted light) respectively, the sum of the two: I ₀ = I _S + I _R is the background light powerful, is the spatially varying phase associated with the sample, and k is the spatial frequency of the carrier fringe.

Then the two interference patterns are subtracted to eliminate the background light intensity, and there is

Perform the Hilbert transform on the above formula to obtain the complex analytical signal related to the sample phase

Among them, HT is the Hilbert transform, and then the complex analysis signal can be expressed as

Z＝HT(I ₊₁ -I _-1 )+j[-(I ₊₁ -I _-1 )] (5)

Among them, j is the imaginary unit. Then the package phase distribution of the sample can be calculated by the following formula

Among them, Im and Re respectively represent the imaginary part and the real part of the complex number. Finally, the purpose of the phase imaging of the present invention can be achieved through the phase unwrapping operation.

Claims (3)

1. a two-step diffraction phase imaging method is characterized in that comprising the following steps: In the first step, the laser beam forms an enlarged microscopic image through a microscopic system, which consists of a sample (6), an adjustable stage (7), a microscopic objective lens (8) and a mirror (9) ; Then the microscopic image passes through the grating (12) on the imaging screen IP behind the microscopic system, thereby being divided into many levels of diffracted light that contains the microscopic image information; In the second step, the diffracted light is subjected to Fourier transform after passing through the third lens (13) behind the imaging screen IP, and on the Fourier plane FP of the third lens (13), utilize the first stop The plate (16) blocks the filter window of the -1st order diffracted light in the spatial light modulator (14), allowing only the +1st order diffracted light to pass through, the 0th order diffracted light is low-pass filtered, and then the +1st order diffracted light and the 0th order diffracted light Carry out Fourier inverse transform by the fourth lens (15) behind described Fourier plane FP, have finished the whole filtering of +1 order diffracted light and 0 order diffracted light, they are respectively as sample light and reference light, in CCD ( 18) Form the first interference pattern on the surface; In the third step, on the Fourier plane FP, the first baffle (16) is removed, and the second baffle (17) is used to block the +1 order diffracted light in the spatial light modulator (14) The filter window allows only -1 order diffracted light and 0 order diffracted light to pass through, and then passes through the fourth lens (15) to form a second interference pattern on the CCD (18). 2. the corresponding phase recovery method of a kind of two-step diffraction phase imaging method according to claim 1, is characterized in that comprising the following steps: The first step is to record two interferograms The interference pattern formed by the 0th order and +1st order diffracted light of the grating is Among them, I _S , I _R are the intensity of the object light wave and the reference light wave respectively, and the sum of the two I ₀ = I _S + I _R is the background light intensity, is the spatially varying phase related to the sample, and k is the spatial frequency of the carrier fringe; the interference pattern formed by the 0-order and -1-order diffracted light of the grating diffraction is The second step is to calculate the difference between the two interference patterns to eliminate the background light intensity The third step is to obtain the complex analysis signal related to the phase First, carry out the Hilbert transform on formula (3) Among them, HT is Hilbert transform; Then the complex analysis signal is Z＝HT(I ₊₁ -I _-1 )+j[-(I ₊₁ -I _-1 )] (5) Among them, j is the imaginary unit; The fourth step is to solve the sample wrapping phase Among them, Im and Re represent the imaginary part and the real part of the complex number respectively; The fifth step is to obtain the real continuous sample phase by unwrapping the formula (6). 3. the corresponding phase recovery method of a kind of two-step diffraction phase imaging method according to claim 2, is characterized in that: utilize the difference of two interferograms to eliminate unnecessary background light intensity, without high-pass filter, to guarantee sample information Integrity, thus suitable for off-axis and slight off-axis interference.