CN107152970B - The parallel microscopic imaging apparatus of high-resolution based on interference array light field - Google Patents

Abstract

The high-resolution parallel microscope imager based on the interference array light field provided by the present invention designs the illumination module to generate four beams of polarized light. Due to the interference of the above four beams of polarized light, an array light field containing a large number of light spots is obtained, thereby realizing the At the same time, the high-resolution parallel microscopic imager based on the interference array light field provided by the present invention uses an area array detector to receive the array spot, and performs pixel redistribution processing on each spot in the array spot to realize Parallel microscopy imaging with high resolution and high signal-to-noise ratio.

Description

High-resolution parallel microscope imager based on interference array light field

technical field

The invention relates to the field of design and manufacture of microscopic detection instruments, in particular to a high-resolution parallel microscopic imager based on an interference array light field.

Background technique

Point-scanning confocal microscopes have been widely used in research fields such as biomedicine, and are indispensable scientific research tools. At present, most commercial point-scanning confocal microscopes use galvanometers for single-point scanning imaging, but the imaging speed of single-point scanning limits the further application of point-scanning confocal microscopy in the field of living cells.

In recent years, many techniques have been proposed to increase the imaging speed of point-scanning confocal microscopy. However, these technologies often sacrifice resolution and other performances to improve imaging speed, and still cannot solve the problem of slow imaging speed of point-scanning confocal microscopy well under the premise of ensuring resolution.

Contents of the invention

The purpose of the present invention is:

Provided is a high-resolution parallel microscopic imager based on an interference array light field with high resolution and fast rendering speed.

To achieve the above object, the present invention adopts the following technical solutions:

A high-resolution parallel microscope imager based on an interference array light field, including an illumination module, a scanning module, a detection module, a control module and an image reconstruction module, wherein:

The illumination module includes a laser, a two-dimensional grating, an aperture, a wave plate, and a lens; the laser is used to emit linearly polarized laser light, and the two-dimensional grating is used to diffract the incident linearly polarized laser light to form two groups of ±1 order diffracted light and 0th order diffracted light, and the polarization direction of the two groups of ±1st order diffracted light and 0th order diffracted light is the same as the polarization direction of the linearly polarized laser light, the diaphragm is used to block the 0th order diffracted light, and Only ±1 diffracted light is allowed to pass through, and the wave plate is used to modulate the incident two sets of ±1 order diffracted light, and rotate the polarization direction of one group of ±1 order diffracted light by 90 degrees, while making the other group ±1 order diffracted light The polarization direction of the diffracted light remains unchanged, and four beams of polarized light whose polarization directions of adjacent beams are perpendicular to each other are obtained, and the four beams of polarized light enter the scanning module after being focused by the lens;

The scanning module includes a dichromatic mirror, an objective lens and a nano-shift stage, the nano-shift stage can move in the XYZ three-dimensional direction, the nano-shift stage carries a sample to be detected, and the four beams of polarized light pass through the dichroic mirror Rear incidence enters the objective lens and interferes at the front focal plane of the objective lens to form an interference array light field with light spots, and the interference array light field with light spots illuminates the sample in parallel, so that the sample Generate parallel optical signals;

The detection module includes a band-pass filter, a detection lens, and an area array detector, and the parallel optical signal enters the band-pass filter, the detection lens, and the area array sequentially after passing through the objective lens and the dichroic mirror. an array detector, and form an array of light spots on the photosensitive surface of the area array detector; allocating processing to form smaller array spots and converting said smaller array spots into electrical signals;

The control module is electrically connected to the area array detector and the nano-displacement stage, and the control module is used to collect the electrical signal;

The image reconstruction module is electrically connected to the control module, and the image reconstruction module collects the electrical signals of the detector and the nano-displacement stage according to the control module to achieve high resolution based on the interference array light field Parallel scan imaging image reconstruction.

In some of these embodiments, the diaphragm is a diaphragm with a solid center.

In some of the embodiments, the area detector is one of CCD or CMOS camera.

The present invention adopts the advantage of above-mentioned technical scheme to be:

The high-resolution parallel microscope imager based on the interference array light field provided by the present invention designs the illumination module to generate four beams of polarized light. Due to the interference of the above four beams of polarized light, an array light field containing a large number of light spots is obtained, thereby realizing the At the same time, the high-resolution parallel microscopic imager based on the interference array light field provided by the present invention uses an area array detector to receive the array spot, and performs pixel redistribution processing on each spot in the array spot to realize Parallel microscopy imaging with high resolution and high signal-to-noise ratio.

The high-resolution parallel microscopic imager based on the interference array light field provided by the present invention utilizes the array light field formed by the interference of four beams of light and the pixel redistribution technology, which can improve the imaging speed and resolution of the point-scanning microscopic technology, and at the same time It can ensure that the image has a high signal-to-noise ratio, improves the image quality, and is conducive to the application of point scanning microscopy in the observation of subcellular structures and the observation of cell dynamic processes, and is beneficial to the development of the biomedical field.

Description of drawings

FIG. 1 is a schematic structural diagram of a high-resolution parallel microscopic imager based on an interference array light field provided by an embodiment of the present invention.

Figure 2(a) is a schematic diagram of the structure of the illumination array spot at the sample;

Fig. 2 (b) is the structural representation of the detection array light spot on the area array detector;

Figure 2(c) is a schematic diagram of the structure of a single array spot and a 1AU virtual pinhole area;

Figure 2(d) is a schematic structural diagram of the pixel area corresponding to the 1AU virtual pinhole;

Fig. 2(e) is a schematic diagram of the structure of the new light spot and the new virtual pinhole after pixel reallocation;

Fig. 2(f) is a schematic structural diagram of the pixel area corresponding to the new virtual pinhole after pixel reallocation;

Figure 2(g) is a schematic diagram of the structure of the new array spot after pixel reallocation;

Figure 3 is a structural schematic diagram of the relative position of the array spot on the sample before (solid) and after moving (dotted line) along the Y direction, the spot spacing is d, and the moving step is _s .

Among them: illumination module 110, scanning module 120, detection module 130, control module 140, image reconstruction module 150, laser 111, two-dimensional grating 112, aperture 113, wave plate 114 and lens 115, dichroic mirror 121, objective lens 122, Nanoshift stage 123 .

Detailed ways

Please refer to FIG. 1, a high-resolution parallel microscope imager 100 based on an interference array light field provided by an embodiment of the present invention, including an illumination module 110, a scanning module 120, a detection module 130, a control module 140 and an image reconstruction module 150 . in:

The illumination module 110 includes a laser 111 , a two-dimensional grating 112 , an aperture 113 , a wave plate 114 and a lens 115 .

Specifically, the laser 111 is used to emit linearly polarized laser light, and the two-dimensional grating is used to diffract the incident linearly polarized laser light to form two groups of ±1st order diffracted light and 0th order diffracted light, and the two groups of ±1 The polarization directions of the first-order diffracted light and the 0-order diffracted light are the same as that of the linearly polarized laser light, the diaphragm is used to block the 0-order diffracted light, and only allows ±1 diffracted light to pass through, and the wave plate is used to modulate Two sets of ±1st-order diffracted lights are incident, and the polarization direction of one group of ±1st-order diffracted lights is rotated by 90 degrees, while the polarization direction of the other group of ±1st-order diffracted lights remains unchanged, and the polarization direction of adjacent beams is obtained Four beams of polarized light perpendicular to each other, the four beams of polarized light are focused by the lens and then enter the scanning module.

Preferably, the diaphragm 113 is a diaphragm with a solid center. It can be understood that the 0th-order diffracted light is blocked by a solid diaphragm with a center, and only ±1 diffracted light is allowed to pass through. Since the distribution direction of the diffracted light is related to the direction of the grating , so the lines connecting the centers of the two groups of ±1 diffracted light beams are perpendicular to each other.

The scanning module 120 includes a dichroic mirror 121 , an objective lens 122 and a nano-stage 123 , the nano-stage 123 can move in the XYZ three-dimensional direction, and the nano-stage 123 carries a sample to be detected.

Specifically, the four beams of polarized light enter the objective lens 122 after passing through the dichroic mirror 121, and interfere at the front focal plane of the objective lens 122 to form an interference array light field with light spots. The interference array light field of light spots illuminates the sample in parallel such that the sample generates parallel light signals.

It can be understood that since the nano-shift stage 123 can move the sample in the XY direction, high-precision parallel scanning and imaging can be realized, and the scanning and imaging speed can be improved.

The detection module 130 includes a bandpass color filter 131 , a detection lens 132 and an area array detector 133 .

Specifically, the parallel optical signal passes through the objective lens 122 and the dichroic mirror 121 and then sequentially enters the bandpass filter 131, the detection lens 132 and the area detector 133, and is detected in the area array. The photosensitive surface of the detector 133 forms an array of light spots, and the area array detector 133 detects the array of light spots, performs spatial filtering on the array of light spots and redistributes the pixels of the array of light spots to form a smaller array spots, and convert the smaller array spots into electrical signals.

Preferably, the area array detector 133 is one of CCD or CMOS camera. It can be understood that the area array detector 133 used in this application is a CCD or CMOS camera with many pixels, which can receive a large number of light spots in the array light field and simultaneously excite the fluorescent signal emitted by the fluorescent sample, and convert it into an electrical signal.

The control module 140 is electrically connected to the area array detector 133, further, the control module 140 is also electrically connected to the nano-displacement stage 123, and the control module 140 is used to control the nano-displacement stage 140 moves.

It can be understood that the control module 140 can realize the scanning control of the nano-displacement stage 123 in the scanning module, and can collect the electrical signal of the area array detector 133 at the same time.

The image reconstruction module 150 is electrically connected to the control module 140, and the image reconstruction module 150 can be implemented according to the electrical signals collected by the control module 140 from the area array detector 133 and the nano-displacement stage 123. High-resolution parallel scanning imaging image reconstruction based on interference array light field.

The working process of the image reconstruction module 150 is described in detail below:

First, the sample is illuminated in parallel by an array light field containing an array of light spots, as shown in Figure 2(a), the sample illuminated by the array light spot will form an array light spot on the photosensitive surface of the area array detector after being imaged by the objective lens and the detection lens (M concentrated light spots) image, as shown in Figure 2(b), realizes the detection and collection of parallel optical signals.

Second, in order to achieve high-resolution imaging, it is necessary to perform spatial filtering and pixel redistribution processing on the detected light spots. Taking the center of each array light spot as the origin, select an Airy disk size (it can be understood that the size of the Airy disk can be determined according to The corresponding pixel area (N pixels) is used as the virtual pinhole of each array spot, as shown in Figure 2(c) and (d); the light intensity I detected by each pixel in the virtual pinhole _i is shifted by s _i /2 (where s _i is the distance from each pixel to the center of the spot) to form a smaller new spot, as shown in Figure 2(e);

Then according to the size of the new spot, re-select a virtual pinhole of a certain size (the area of the virtual pinhole is taken as an example of the size of the new spot, as shown in the smaller dashed box in Figure 2(e) and Figure 2(f), Corresponding to Q pixels in the area array detector), the light intensity _k detected by each pixel in the virtual pinhole is superimposed, and the pixel value of the high-resolution microscopic image corresponding to a point in the sample can be obtained This process is the pixel reprocessing process.

Third, redistribute the pixels of each array spot on the area array detector to form smaller array spots (as shown in Figure 2(g), the black dotted line is the original array spot, and the solid spot is the small new one Array spot) to obtain the high-resolution microscopic image pixel values corresponding to multiple points in the sample. After all spot processing and calculations, a high-resolution microscopic image with pixel values at intervals will be obtained.

Fourth, when the sample is scanned, as shown in Figure 3, the array spot on the sample moves (the dotted line is the moved array spot), the position of the array spot on the array detector remains unchanged, and the spot in the array light field irradiating the sample The distance is d, if the displacement stage is moved along the Y direction, the movement distance is _s , and a high-resolution microscopic image with pixel values at intervals will be obtained every time it is moved, and d/d _s times of movement can obtain d/d _s pixels If the high-resolution microscopic images distributed at intervals of pixel values, if the displacement stage is moved d/d _s -1 times along the X direction, d/d _s high-resolution microscopic images can also be obtained, and the d/d _s pixel value intervals The distributed high-resolution microscopic images are superimposed to obtain a complete high-resolution microscopic image.

The working process of the high-resolution parallel microscopic imager 100 based on the interference array light field provided by the present invention is as follows:

A beam of linearly polarized laser light emitted by the laser 111 passes through the two-dimensional grating 112, the aperture 113, the wave plate 114, and the lens 115 in sequence to form four polarized beams whose polarization directions of adjacent beams are perpendicular to each other. Light, the four beams of polarized light enter the objective lens 122 after passing through the dichroic mirror 121, and interfere at the front focal plane of the objective lens 122 to form an interference array light field with light spots. The interference array light field illuminates the sample in parallel, so that the sample generates parallel light signals, and the parallel light signals pass through the objective lens 122 and the dichroic mirror 121 and then enter the bandpass filter in sequence 131. Detecting the lens 132 and the area array detector 133, and forming an array of light spots on the photosensitive surface of the area array detector 133, the area array detector 133 detects the array of light spots, and performs spatial filtering on the array of light spots and redistribute the pixels of the array light spots to form smaller array light spots, and at the same time convert the array light spots into electrical signals, the control module 140 collects the electrical signals, and the image reconstruction module 150 Collecting the electrical signal according to the control module 140 implements high-resolution parallel scanning imaging image reconstruction based on the interference array light field.

The high-resolution parallel microscope imager 100 based on the interference array light field provided by the present invention designs the illumination module 110 to generate four beams of polarized light. Since the above-mentioned four beams of polarized light interfere to obtain an array light field containing a large number of light spots, thereby realizing At the same time, the high-resolution parallel microscopic imager based on the interference array light field provided by the present invention adopts the area array detector 133 to receive the array light spots, and redistributes the pixels of each light spot in the array light spot Processing to achieve parallel microscopic imaging with high resolution and high signal-to-noise ratio.

The high-resolution parallel microscopic imager based on the interference array light field provided by the present invention utilizes the array light field formed by the interference of four beams of light and the pixel redistribution technology, which can improve the imaging speed and resolution of the point-scanning microscopic technology, and at the same time It can ensure that the image has a high signal-to-noise ratio, improves the image quality, and is conducive to the application of point scanning microscopy in the observation of subcellular structures and the observation of cell dynamic processes, and is beneficial to the development of the biomedical field.

Of course, the high-resolution parallel microscopic imager based on the interference array light field of the present invention can also have various transformations and modifications, and is not limited to the specific structure of the above-mentioned embodiment. In a word, the protection scope of the present invention shall include those transformations, substitutions and modifications obvious to those skilled in the art.

Claims (3)

1. A high-resolution parallel microscopic imager based on an interference array light field, characterized in that it includes an illumination module, a scanning module, a detection module, a control module and an image reconstruction module, wherein: The illumination module includes a laser, a two-dimensional grating, an aperture, a wave plate, and a lens; the laser is used to emit linearly polarized laser light, and the two-dimensional grating is used to diffract the incident linearly polarized laser light to form two groups of ±1 order diffracted light and 0th order diffracted light, and the polarization direction of the two groups of ±1st order diffracted light and 0th order diffracted light is the same as the polarization direction of the linearly polarized laser light, the diaphragm is used to block the 0th order diffracted light, and Only the ±1st-order diffracted light is allowed to pass through, and the wave plate is used to modulate the incident two sets of ±1st-order diffracted lights, and rotate the polarization direction of one set of ±1st-order diffracted lights by 90 degrees, while making the other set of ±1st-order diffracted lights The polarization direction of the first-order diffracted light remains unchanged, and four beams of polarized light whose polarization directions of adjacent beams are perpendicular to each other are obtained, and the four beams of polarized light enter the scanning module after being focused by the lens; The scanning module includes a dichromatic mirror, an objective lens and a nano-shift stage, the nano-shift stage can move in the XYZ three-dimensional direction, the nano-shift stage carries a sample to be detected, and the four beams of polarized light pass through the dichroic mirror Rear incidence enters the objective lens and interferes at the front focal plane of the objective lens to form an interference array light field with light spots, and the interference array light field with light spots illuminates the sample in parallel, so that the sample Generate parallel optical signals; The detection module includes a band-pass filter, a detection lens, and an area array detector, and the parallel optical signal enters the band-pass filter, the detection lens, and the area array sequentially after passing through the objective lens and the dichroic mirror. an array detector, and form an array of light spots on the photosensitive surface of the area array detector; allocating processing to form smaller array spots and converting said smaller array spots into electrical signals; The control module is electrically connected to the area array detector and the nano-displacement stage, and the control module is used to collect the electrical signal; The image reconstruction module is electrically connected to the control module, and the image reconstruction module collects the electrical signals of the detector and the nano-displacement stage according to the control module to achieve high resolution based on the interference array light field Parallel scan imaging image reconstruction. 2 . The high-resolution parallel microscope imager based on interference array light field according to claim 1 , wherein the aperture is a solid aperture at the center. 3 . 3. The high-resolution parallel microscope imager based on the interference array light field according to claim 1, wherein the area array detector is one of a CCD or a CMOS camera.