Patent Description:
In the technical field of optical imaging, an out-of-focus background interference of a traditional wide-field microscope makes it impossible to obtain a sharp image of the focal plane. Generally, the background interference can be avoided by cutting a tissue into slices. Optical slicing can achieve an imaging effect similar to that of the tissue slicing by an optical imaging method, and can also be referred to as optical sectioning. Confocal microscopic imaging technology can block a defocusing background interference and only allow the passage of an effective signal of the focal plane by placing a pinhole in front of a detector, thereby achieving an optical sectioning effect. Multi-photon excitation microscopic imaging technology has enough energy to excite fluorescence signal only at a focal point of a sample by utilizing a nonlinear effect, thereby achieving an ideal optical sectioning effect. However, both of the two optical sectioning technologies adopt a point-by-point scanning imaging mode which has an obviously insufficient imaging throughput in comparison with the wide-field imaging mode.

Structured illumination microscopic imaging technology implements modulation of a focal plane signal by superimposing a high-frequency periodic pattern modulation on a wide-field illumination, and a defocusing signal is suppressed due to rapid attenuation of the high-frequency modulation, thereby achieving optical sectioning. In the implementation of this process, at least three original images with different modulation phases are required, and the focal plane signal is demodulated by using a structured illumination microscopic imaging reconstruction algorithm to obtain an optical sectioning image. Compared with the confocal and multi-photon excitation microscopic imaging technologies which also have an optical sectioning ability respectively, the structured illumination microscopic imaging has advantages of high imaging throughput due to the wide-field imaging manner. When a large-size sample needs to be imaged, the structured illumination microscopic imaging technology generally needs to use a mosaic stitching method to expand the imaging field. In this way, most of the time spent for imaging the large-size sample is used for movement of the sample between the mosaics, therefore the overall imaging speed is limited. In order to avoid an excessive mosaic stitching, <CIT> discloses a structured light fast scan imaging method which uses line scanning and strip imaging to improve the imaging speed, and uses structured illumination to suppress the background interference, thereby realizing acquiring an optical sectioning image of a large-size sample quickly. However, this method also needs to scan back and forth the imaging area of the sample three times to obtain raw data required for reconstruction of a structured illumination microscopic optical sectioning image, and therefore sacrifices the imaging speed. In addition, this imaging method needs to use a light beam modulation device in a strip imaging system to achieve modulation of the illumination light field, thereby increasing the complexity of the system. Meanwhile, because it uses a conventional structured illumination microscopic imaging method, imaging quality is highly dependent on the contrast of the modulation pattern. <NPL> (DOI: <NUM>/OL. <NUM>) also discloses a related method of optical sectioning microscopy.

Therefore, it is necessary to develop a simple and efficient high-throughput optical sectioning imaging method and imaging system.

An object of the present disclosure is to overcome the above technical deficiencies, propose a high-throughput optical sectioning imaging method and imaging system, and solve the technical problems of the structured illumination microscopic imaging technology in the prior art having a low imaging speed of a large-size sample, requiring additional modulation elements, being highly dependent on the contrast of the modulation pattern, and having a complex demodulation algorithm for reconstruction of an optical sectioning image.

To achieve the above technical object, the technical solution of the present disclosure provides a high-throughput optical sectioning imaging method which includes the following steps:.

Meanwhile, the present disclosure also provides a high-throughput optical sectioning imaging system which includes:.

Compared with the prior art, the present disclosure performs illumination by a light beam having incompletely identical modulated intensities, images a same sample in different rows of pixels, and obtain a focal plane image by using a simpler demodulation algorithm, which simplifies a structured illumination microscopic reconstruction algorithm, improves reconstruction efficiency, and improves an imaging speed of large-size samples.

In order to make objects, technical solutions, and advantages of the present disclosure more apparent, the present disclosure will be further described in detail below with reference to the accompanying drawings and embodiments. It should be appreciated that the specific embodiments described herein are merely intended to explain the present disclosure and are not intended to limit the present disclosure.

As shown in <FIG>, the present disclosure provides a high-throughput optical sectioning imaging method which includes the following steps.

At S1, a light beam is modulated into a modulated light beam capable of being focused on a focal plane of an objective lens and capable of being defocused on a defocusing plane of the objective lens, the modulated light beam having incompletely identical modulated intensities on the focal plane of the objective lens.

Particularly, when modulated, a light beam is firstly shaped into a linear light beam. Then, the linear light beam is modulated into a modulated light beam for linear light illumination. This embodiment allows a sample to be illuminated by a linear modulated light beam capable of being focused on a focal plane of an objective lens and being defocused on a defocusing plane of the objective lens, which can facilitate exciting the sample to emit fluorescence, thereby facilitating subsequent imaging.

Here, the above-mentioned modulated light beam in the focal plane of the objective lens has been specifically subject to a waveform modulation with incompletely identical modulation intensities, for example, Gaussian modulation, sinusoidal modulation, or triangular modulation or the like with incompletely identical modulation intensities. Since the illumination light beam of this embodiment adopts a Gaussian beam, the modulated light beam for light illumination formed in this embodiment is formed by the Gaussian modulation. This embodiment may also use other waveform modulations with incompletely identical modulation intensities as needed.

At S2, a sample under illumination of the modulated light beam is imaged in different rows of pixels to form sample images, a formula expression of the formed sample image being <MAT>
where I(i) is a sample image formed in an ith row of pixels, f(i) is a modulation intensity corresponding to the sample image I(i), Iin is a focal plane image of the sample image, and Iout is a defocusing plane image of the sample image.

When imaging, step S2 particularly includes the following steps.

At S21, the modulated light beam and the sample are driven to make a relative movement to each other continuously at a constant speed in the X direction.

At S22, the sample is imaged, by a camera, along a direction of the relative movement continuously and sequentially.

In this embodiment, the modulated light beam may be perpendicular to a direction along which the sample moves, and a direction along which the imaging of the sample is performed continuously is the same as a direction along which multiple rows of pixels are arrayed. That is to say, during the process of the relative movement between the sample and the modulated light beam, a part subject to continuous illumination of the sample is imaged continuously. Here, in this embodiment, it can drive the sample to move continuously at a constant speed along a direction perpendicular to the modulated light beam for linear illumination, or it can drive the modulated light beam to move continuously at a constant speed along a direction parallel to the sample, provided that there is a continuous and constant speed relative movement between the modulated light beam and the sample.

As shown in (a) of <FIG>, an imaging area in this embodiment has N rows of pixels, where N ≥ <NUM>. Two directions X and Y perpendicular to each other are formed on a plane parallel to an imaging plane of the sample. The modulated light beam has following characteristics in the X and Y directions respectively: the modulated light beam has incompletely identical modulated intensities along the X direction on the N rows of pixels, and the modulated light beam has the same modulated intensity along the Y direction on each row of the N rows of pixels. Furthermore, a distribution direction and width of the N rows of pixels are the same as and in an object-image conjugate relationship with a distribution direction and width of the modulated light beam for linear light illumination respectively, facilitating the correspondence of the imaging area to the modulated light beam for linear light illumination.

Correspondingly, the sample moves relative to the modulated light beam along the X direction, so as to ensure that the direction along which the relative movement between the modulated light beam and the sample is performed is the same as a direction along which the N rows of pixels are arrayed. For ease of operation, as a preferred example of this embodiment, the sample is driven to move and the modulated light beam is set to be stationary. In this case, a movement direction of the sample is set to be the same as the direction along which the N rows of pixels are arrayed, and a single frame exposure duration for imaging is equal to a duration spent by the sample moving by one row of pixels. If an image corresponding to any row of pixels in one image frame is defined as one strip image block, multiple strip image blocks corresponding to any row of pixels in multiple image frames are formed by continuous and sequential imaging of each part of the sample and may be stitched into one strip image, and the N rows of pixels may form N strip images.

Here, in this embodiment, the status of imaging can be determined. When it is determined that continuous imaging is completed, subsequent steps may be performed; and when it is determined that continuous imaging is not completed, the sample is continuously driven to move. In this embodiment, continuous imaging of the sample is realized by continuous and constant speed movement of the sample and thus is equivalent to continuous scanning imaging of the sample. Therefore after the imaging, it needs to determine whether the continuous scanning imaging of the whole sample is completed, which facilitates ensuring integrity and continuity of the imaging.

At S23, a strip image block It(i) of an ith row of pixels in each image frame obtained in an chronological order is acquired, the strip image block being expressed by the formula: <MAT>
where It(i) is a strip image block corresponding to the ith row of pixels in the tth image frame, <MAT> is a focal plane image of the strip image block corresponding to It(i), that is, <MAT> is a focal plane image of the mth strip image block in a complete strip image, <MAT> is a defocusing image of the strip image block corresponding to It(i), and f(i) is a modulation intensity corresponding to the ith row of pixels.

As shown in (a) of <FIG>, when imaged, the sample moves in the direction along which the imaging pixels are arrayed. Since the single frame exposure duration for imaging is equal to the duration spent by the sample moving by one row of pixels, each row of pixels sequentially form a plurality of strip image blocks along a lengthwise direction of a sample strip which are formed by continuous imaging of the sample.

At S24, strip image blocks of the ith row of pixels in each image frame are stitched successively to obtain a strip image of the ith row of pixels, a formula expression of the strip image being: <MAT>
where M is a number of strip image blocks corresponding to the complete strip image, and specifically, the strip image is formed by stitching M strip image blocks, where <MAT> is a focal plane image corresponding to the mth strip image block in the strip image, and m≤M.

It should be noted that, the strip image is formed by shifting and stitching a plurality of strip image blocks corresponding to a row of pixels, that is, strip image blocks of N rows of pixels may be respectively stitched to form N strip images.

At S3, focal plane images (i.e., optical sectioning images) of strip images in the different rows of pixels are obtained by demodulating the strip images according to a demodulation algorithm, the demodulation formula of the demodulation algorithm being <MAT>
where α and β are positive integers, c is a constant greater than <NUM>, I<NUM> is an accumulated sum of strip images acquired in α pixels, and I<NUM> is an accumulated sum of sample images acquired in β pixels; an accumulated value of modulation intensities corresponding to the sample images in the α pixels is different from an accumulated value of modulation intensities corresponding to the sample images in the β pixels.

The step S3 particularly includes following steps.

At S31, strip images of at least one row of pixels are accumulated to form a first strip image, and strip images of at least one row of pixels are accumulated to form a second strip image.

When the N strip images are acquired, one or two or more of the strip images may be arbitrarily selected to accumulate and form the first strip image. Then, the second strip image is obtained by accumulation in the same manner. In order to avoid that the optical sectioning image acquired by the above demodulation algorithm is zero, in this embodiment, an accumulated value of the modulation intensities corresponding to the strip images in α pixels may be different from an accumulated value of the modulation intensities corresponding to the strip images in β pixels.

At S32, the first strip image and the second strip image are demodulated into an optical sectioning image of the strip image according to the demodulation formula. Then we get <MAT>.

For the convenience of explanation of the acquisition process of the strip image of this embodiment, the following embodiments will be described.

Embodiment <NUM>: As shown in (a) of <FIG>, when the sample moves in the direction along which N rows of pixels are arrayed, N+M-<NUM> image frames can be obtained within a time interval from time t<NUM> to tN+M-<NUM> (M is the number of strip image blocks corresponding to a complete strip image, N is <NUM> and M is <NUM> in this embodiment). In addition, each row of pixels in the N+M-<NUM> image frames corresponds to a strip image block. For example, a strip image block I<NUM>(<NUM>) of a first row of pixels in a first image frame, a strip image block I<NUM>(<NUM>) of the first row of pixels of a second image frame, a strip image block IN(<NUM>) of the first row of pixels of the Nth image frame, and a strip image block IN+M-<NUM>(<NUM>) of the first row of pixels of the (N+M-<NUM>)tn image frame can be obtained. The strip image block I<NUM>(<NUM>), the strip image block I<NUM>(<NUM>) to the strip image block IN+M-<NUM>(<NUM>) may be successively stitched to form a strip image, and each of corresponding second to Nth rows of pixels may be stitched to form a corresponding strip image.

As shown in (b) and (c) of <FIG>, in order to explain how to acquire a clearer strip image block and a clearer strip image, firstly, the second row of pixels and the fourth row of pixels are taken as examples for description. Because <NUM>(<NUM>) = <MAT> and <MAT> can be obtained from the formulas of the strip image block and the sample image respectively, the strip image block in the fourth row of pixels of the fourth image frame is <MAT> (where m = <NUM>, because a strip image is formed by stitching nine strip image blocks, and the strip image block in the fourth row of pixels in the fourth image frame is the first strip image block of the strip image, that is, <MAT> is a focal plane image corresponding to a first strip image block in the strip image). Correspondingly, <MAT>, where <MAT>, the strip image block in the second row of pixels of the second image frame is <MAT>; I<NUM> is an accumulated sum of the sample images acquired in the fourth row of pixels, that is <MAT>, I<NUM> is an accumulated sum of the sample images acquired in the second row of pixels, that is <MAT>, the values of α and β are both selected as <NUM>. I <NUM>(<NUM>) - I(<NUM>)| = | <MAT>, therefore <MAT> <MAT>.

Embodiment <NUM>: as shown in <FIG>, the strip image formed by stitching in the fourth row of pixels is <MAT>, where <MAT>; the strip image formed by stitching in the first row of pixels is <MAT>, where <MAT>; the strip image formed by stitching in the second row of pixels is <MAT>, where <MAT>; and the strip image formed by stitching in the third row of pixels is <MAT>, where It(<NUM>) = <MAT>.

If I<NUM> is an accumulated sum of the sample images acquired in the first, second and third rows of pixels, that is <MAT>, and I<NUM> is an accumulated sum of the sample images acquired in the fourth row of pixels, that is <MAT>, correspondingly, the value of α should be selected as <NUM>, and the value of β should be selected as <NUM>. I (<NUM>(<NUM>) + <NUM>(<NUM>) + I(<NUM>)) - <NUM>(<NUM>) I = I <MAT> <MAT> can be obtained from the demodulation formula, therefore <MAT>.

For convenience of illustrating this embodiment, as shown in <FIG>, this embodiment also provides a high-throughput optical sectioning imaging system <NUM> including a light beam modulation module <NUM>, an imaging module <NUM> and a demodulation module <NUM>.

The light beam modulation module <NUM> is configured to modulate a light beam into a modulated light beam capable of being focused on a focal plane of an objective lens and capable of being defocused on a defocusing plane of the objective lens, and the modulated light beam has incompletely identical modulated intensities on the focal plane of the objective lens.

The light beam modulation module <NUM> in this embodiment includes a shaping optical path for shaping illumination light into a linear light beam and a modulation optical path for modulating the linear light beam into a modulated light beam for linear light illumination. The shaping optical path includes a laser light source <NUM>, a first lens <NUM>, a second lens <NUM> and a cylindrical lens <NUM> which are sequentially arranged along a travel direction of the illumination light. The modulation optical path includes a third lens <NUM> configured to modulate divergent light of the linear light beam into parallel light, a dichroic mirror <NUM> configured to modulate an incident direction of the linear light beam, and an objective lens <NUM> arranged coaxially with the linear light beam the incident direction of which has been modulated.

During the light modulation, the laser light source <NUM> emits illumination light which is sequentially processed by the first lens <NUM> and the second lens <NUM> so as to be an expanded light beam. The expanded light beam is shaped by the cylindrical lens <NUM> to form a linear light beam 11a. The linear light beam 11a is a divergent light. Then, the linear light beam 11a forms the parallel light rays after passing through the third lens <NUM>. Then, the dichroic mirror <NUM> changes an incident direction of the line light beam 11a, and then the linear light beam 11a enters the objective lens <NUM> to form a modulated linear light beam 11b which is capable of being focused on the focal plane of the objective lens <NUM> and capable of being defocused on a defocusing plane of the objective lens <NUM>. In order to facilitate subsequent imaging, an optical axis of the modulated linear light beam 11b is perpendicular to an optical axis of the illumination light and an optical axis of the linear light beam 11a which has not been reflected, that is, the first lens <NUM>, the second lens <NUM>, the cylindrical lens <NUM> and the third lens <NUM> are arranged coaxially, and central axes of the first lens <NUM>, the second lens <NUM>, the cylindrical lens <NUM> and the third lens <NUM> are arranged perpendicular to a central axis of the objective lens <NUM>. Furthermore, the angle between the dichroic mirror <NUM> and the optical axis of the modulated light beam 11b for linear illumination is <NUM> degrees, ensuring that the width of the linear light beam 11a after being reflected by the dichroic mirror <NUM> does not change.

The imaging module <NUM> is configured to employ a camera to image, in different rows of pixels, a same sample under illumination of the modulated light beam. The imaging module <NUM> includes a driving unit <NUM>, an imaging unit <NUM>, an image block acquisition unit <NUM> and a stitching unit <NUM>. A formula expression of a sample image formed by imaging using the imaging module <NUM> is I(i) = Iinf(i) + Iout, where I(i) is a sample image formed in the ith row of pixels, f(i) is a modulation intensity corresponding to the sample image I(i), Iin is a focal plane image of the sample image, and Iout is a defocusing plane image of the sample image.

The driving unit <NUM> is configured to drive the modulated light beam 11b and the sample <NUM> to make a relative movement to each other continuously at a constant speed along the X direction, and a single frame exposure duration in the camera is equal to a duration spent by the relative movement by one row of pixels. In order to facilitate the driving, the driving unit <NUM> in this embodiment may adopt a translation stage which can drive the sample <NUM> to move continuously at a constant speed along a direction perpendicular to the modulated light beam 11b. The translation stage <NUM> may be a motorized translation stage <NUM> and may be located directly below the objective lens <NUM>. The sample <NUM> is provided on the translation stage <NUM> and can move along with the translation stage <NUM>. In order to control imaging precision, an upper surface of the translation stage <NUM> is perpendicular to the optical axis of the modulated linear light beam 11b. The sample <NUM> is arranged on the translation stage <NUM> and passes through a modulated region of the modulated linear light beam 11b during the process of movement. Under the effect of the modulated linear light beam 11b, the sample <NUM> is excited to emit fluorescence. The translation stage <NUM> in this embodiment is in a horizontal state, and the modulated linear light beam 11b is parallel with the translation stage and is perpendicular to the movement direction of the sample <NUM>.

The imaging unit <NUM> is configured to perform successive imaging along the direction in which the sample <NUM> performs the above relative movement. Particularly, the imaging unit <NUM> is configured to perform successive and continuous imaging as the sample <NUM> moves continuously, which can be realized by an imaging optical path. The imaging optical path is composed of an emission filter 122a, a tube lens 122b and a camera 122c which are located directly above the objective lens <NUM>. The fluorescence from the excited sample <NUM> passes through the objective lens <NUM>, the dichroic mirror <NUM>, the emission filter 122a and the tube lens 122b sequentially, and then is detected and imaged by the camera 122c. Here, the camera 122c of this embodiment may be a planar array Charge-coupled device (CCD) or planar array Complementary Metal Oxide Semiconductor (CMOS) camera having a function of Sub-array or Region of interest (ROI), or may be a linear array CCD or linear array CMOS camera having an array mode. In order to facilitate subsequent reconstruction of an optical sectioning image, an imaging area of the camera 122c in this embodiment has N rows of pixels, where N ≥ <NUM>, and the imaging direction of the camera 122c and the width of the imaging area are the same as the direction and width of the modulated light beam 11b for linear light illumination, respectively. A single frame exposure duration of the camera 122c is equal to a duration spent by the translation stage driving the sample <NUM> to move by one row of pixels, which is described above and is omitted here.

The image block acquisition unit <NUM> is configured to acquire a strip image block of an ith row of pixels in each image frame obtained in an chronological order, and the strip image block is expressed by the formula: <MAT>
where It(i) is a strip image block corresponding to the ith row of pixels in the tth image frame , <MAT> is a focal plane image of the strip image block corresponding to It(i), that is, <MAT> is a focal plane image of the mth strip image block in a complete strip image, <MAT> is a defocusing image of the strip image block corresponding to It(i), and f(i) is a modulation intensity corresponding to the ith row of pixels.

The stitching unit <NUM> is configured to successively stitch strip image blocks of the ith row of pixels in each image frame to obtain a strip image of the ith row of pixels according to the formula of <MAT>It(i), where M is a number of strip image blocks corresponding to the complete strip image, and specifically, the strip image is formed by stitching M strip image blocks, where <MAT> is a focal plane image corresponding to the mth strip image block in the strip image, and m≤M.

The demodulation module <NUM> is configured to demodulate multiple sample images using the demodulation algorithm to obtain focal plane images of the multiple sample images. The demodulation module <NUM> may include an image accumulation unit <NUM> and a demodulation unit <NUM>. The sample image in this embodiment is a strip image. Therefore, The image accumulation unit <NUM> is configured to accumulate strip images of at least one row of pixels to form a first strip image, and accumulate strip images of at least one row of pixels to form a second strip image. The demodulation unit <NUM> is configured to demodulate the first strip image and the second strip image into optical sectioning images of the strip images according to the demodulation algorithm. It should be noted that, the focal plane image in this embodiment is an optical sectioning image. Here, the demodulation formula of the demodulation algorithm is Iin = c × | β I<NUM>- α I<NUM> |, where α and β are positive integers, c is a constant greater than <NUM>, I<NUM> is an accumulated sum of strip images acquired in α pixels, and I<NUM> is an accumulated sum of sample images acquired in β pixels; an accumulated value of modulation intensities corresponding to the sample images in the α pixels is different from an accumulated value of modulation intensities corresponding to the sample images in the β pixels.

Specific functions and actions of the image block acquisition unit <NUM>, the stitching unit <NUM>, the image accumulation unit <NUM> and the demodulation unit <NUM> have been described in detail in the above.

Claim 1:
A high-throughput optical sectioning imaging method, comprising the following steps:
at S1, modulating a light beam into a modulated light beam (11b) capable of being focused on a focal plane of an objective lens (<NUM>) and being defocused on a defocusing plane of the objective lens (<NUM>);
at S2, imaging, by a camera (122c), in different rows of pixels, a same sample (<NUM>) under illumination of the modulated light beam (11b) to form sample images, wherein the modulated light beam (11b) has incompletely identical modulated intensities on the different rows of pixels in a direction along which the different rows of pixels are arrayed, the sample (<NUM>) moves in the direction along which the different rows of pixels are arrayed when imaged, and each of the different rows of pixels sequentially forms a plurality of strip image blocks in the direction along which the different rows of pixels are arrayed, the plurality of strip image blocks formed by each of the different rows of pixels are used to be stitched into a corresponding one of the sample images of the same sample (<NUM>) and a formula expression of the formed sample image is: <MAT>
where I(i) is a sample image formed in an ith row of pixels, f(i) is a modulation intensity corresponding to the sample image I(i), Iin is a focal plane image of the sample image, and Iout is a defocusing plane image of the sample image;
at S3, obtaining focal plane images of the sample images in the different rows of pixels by demodulating the sample images according to a demodulation algorithm, the focal plane image being an optical sectioning image, and a demodulation formula of the demodulation algorithm being Iin = c × | β I<NUM> - α I<NUM> |,
where α and β are positive integers, c is a constant greater than <NUM>, I<NUM> is an accumulated sum of sample images acquired in α rows of pixels, and I<NUM> is an accumulated sum of sample images acquired in β rows of pixels; β × (an accumulated value of modulation intensities corresponding to the sample images in the α rows of pixels) is different from α × (an accumulated value of modulation intensities corresponding to the sample images in the β rows of pixels).