CN105699276B - A kind of super-resolution imaging device and method - Google Patents

Abstract

The invention discloses a device and method for super-resolution microscopic imaging. The device comprises a blue LED light source, a convex lens L ₁ , a low-pass filter O1, a dichroic prism, an encoder, a high-pass filter O2, a convex lens L ₂ , CCD camera and computer, the blue light emitted by the blue LED light source sequentially passes through the convex lens _L1 , low-pass filter O1, beam splitter, encoder, and irradiates the sample to be tested. Prism, high-pass filter O2, and convex lens _L2 are sent to the computer by the CCD camera. The encoder is a random nano particle encoder. The microscopic device and its method are characterized in that the principle of traditional optical microscope is adopted, the decoding is completely realized by computer, and the structure is simple; the light source adopts LED light source to excite the sample, no laser irradiation is needed, and the degree of damage to living cells is small.

Description

A super-resolution imaging device and method

technical field

The invention relates to a microscopic imaging device and method, in particular to a device and method for microscopic imaging of living cells by breaking through the diffraction limit of an optical microscope and achieving nanoscale resolution.

Background technique

The development of modern biology has put forward higher and higher resolution requirements for microstructure research, hoping to reveal the physical essence of life processes from the molecular level. However, limited by the optical diffraction limit, the lateral resolution of ordinary optical microscopes can only reach 200nm and the vertical resolution can reach 500nm, which makes it impossible to study the subcellular structure and the interaction of its internal substances. Although electron microscopy and atomic force microscopy can achieve nanometer-level resolution, their shortcomings in the observation of non-viable isolated cell samples limit their wide application in the biological field. Therefore, how to break through the resolution limit of traditional optical microscopes and perform nanoscale microscopic imaging of living cells has become an urgent problem to be solved in optical microscopic imaging technology.

In recent years, with the continuous development of computers and optical devices, super-resolution microscopic imaging techniques that can break through the diffraction limit of traditional optical microscopes have emerged one after another, solving the shortcomings of electron microscopes and atomic force microscopes that cannot observe living isolated cell samples. Realize microscopic imaging with 1-100nm resolution. Its core idea is to obtain a high spatial resolution image by establishing time bandwidth information, which is specifically expressed as obtaining a high spatial resolution image from a time series of low spatial resolution images. Commonly used super-resolution techniques mainly include the following methods: 1) 4 Pi microscopy: This technique can achieve nanometer resolution and in vivo measurement, but requires the use of interference methods and the complex structure of dual objective lenses; 2) Stimulated emission depletion microscopy Technology: This technology can break through the far-field optical microscopy of optical diffraction to obtain super-resolution images, but this technology requires the use of supercontinuum lasers and long-term laser irradiation will destroy living cells; 3) Light-activated localization microscopy technology: this technology Molecular activities at the nanometer level can be observed, but it requires specific wavelengths of light, repeated excitation and bleaching of fluorescent molecules, complex structures, and extremely low measurement efficiency.

Contents of the invention

The object of the present invention is to provide a super-resolution imaging device. The device of the present invention is characterized by a simple structure, an LED light source is used as a light source, and little damage to observed living cells.

Another object of the present invention is a method for imaging with a super-resolution imaging device.

To achieve the above object, the technical solution adopted in the present invention is:

A device for super-resolution microscopic imaging, including a blue LED light source, a convex lens L ₁ , a low-pass filter O1, a spectroscopic prism, an encoder, a high-pass filter O2, a convex lens L ₂ , a CCD camera and a computer, and the blue light emitted by the LED light source The blue light passes through the convex lens L ₁ , the low-pass filter O1, the dichroic prism, and the encoder in sequence, and irradiates the sample to be tested. ₂ , and then sent to the computer by the CCD camera.

Further, the encoder is a random nano particle encoder.

A method for performing microscopic imaging using a super-resolution microscopic imaging device, characterized in that: comprising the following steps:

1), the blue light emitted by the blue LED light source is collimated by the convex lens _L1 , the low-pass filter O1, and the dichroic prism excite the tested sample to generate emitted light s ( x , y );

2) The encoder generates a time-varying random code f ( x , y , t ) through nanoparticles, and encodes the emitted light s ( x , y ) of the sample to be tested. After passing through the beam splitter prism, high-pass filter O2 and convex lens L ₂ and then focus on the image plane of the CCD camera;

3) The computer generates time-varying decoding f´ ( x , y , t ) according to the time-varying random code:

,

Among them, c is a constant, δ ( x , y ) is a two-dimensional unit pulse function;

4) Time-varying decoding of the images collected by the CCD camera is as follows:

,

In the formula, p ( x , y ) is the point spread function of the convex lens L ₂ ;

5) Reconstruct the time-varying decoded image o ₂ ( x , y , t ) as follows:

In the above formula, p (0 , 0) is the value of the center point of the point spread function, and o ( x , y ) is the super-resolution reconstructed image of the original image s ( x , y ).

Beneficial effects of the present invention:

The random coding super-resolution imaging microscopic device and its method proposed by the present invention are characterized in that the principle of traditional optical microscope is adopted, the decoding is completely realized by computer, and the structure is simple; The degree of damage is small.

Description of drawings

Below in conjunction with accompanying drawing and specific embodiment the present invention is described in further detail:

Fig. 1 is a structural representation of the present invention;

Figure 2 is a distribution diagram of the original image information of the tested sample;

Figure 3 is a distribution diagram of random nanoparticle codes;

Figure 4 is a super-resolution image decoded by a computer;

Fig. 5 is a comparison and combination diagram of the original image, blurred image and super-resolution image profile.

In the figure: 1. Blue LED light source; 2. Convex lens L ₁ ; 3. Low-pass filter O1; 4. Dichroic prism; 5. Encoder; 6. Tested sample; 7. High-pass filter O2; 8. Convex lens L ₂ ; 9. CCD camera; 10. Computer.

Detailed ways

As shown in Figure 1, a device for super-resolution microscopic imaging includes a blue light LED light source 1, a convex lens L ₁ 2, a low-pass filter O13, a dichroic prism 4, an encoder 5, a high-pass filter O27, and a convex lens L _28. CCD camera 9 and computer 10, the blue light emitted by blue LED light source 1 passes through convex lens L ₁ 2, low-pass filter O13, dichroic prism 4, and encoder 5 in sequence, and then irradiates on the tested sample 6, and the tested sample The emitted light generated by 6 passes through the encoder 5, the dichroic prism 4, the high-pass filter O27, the convex lens _L28 in turn, and then is sent to the computer 10 by the CCD camera 9.

The encoder 5 is encoded by random nanoparticles.

A method for performing microscopic imaging using a super-resolution microscopic imaging device, characterized in that: comprising the following steps:

1) Illumination light path: Luxem Ⅲ Royal blue blue light LED light source 1 emits blue light with a wavelength range of 440nm~460nm, which is collimated by a convex lens L ₁ 2 with a focal length of f _L1 =60mm and becomes parallel light. Filtered by the 470nm low-pass filter O13, and finally excited the measured sample 6 through the 50:50 dichroic prism 4 to generate the sample emission light s ( x , y );

2), Imaging optical path part: the nanoparticle encoder 5 generates a time-varying random code f ( x , y , t ), randomly encodes the emitted light s ( x , y ) of the sample 6 under test, and the coded sample emits light And after the blue-light excitation light passes through the dichroic prism 4 and the 470nm high-pass filter O27, the blue-light excitation light is filtered out to obtain information containing only the light emitted by the sample. Computer-simulated distribution of the original image information of the tested sample (as shown in Figure 2); computer-simulated distribution of time-varying random nanoparticle codes (as shown in Figure 3). The emitted light of the tested sample is focused on the CCD9 image plane with a pixel size of 1392×1040 through a convex lens L ₂ 8 with a focal length of f _L2 =150mm;

3) The computer generates time-varying decoding f´ ( x , y , t ) according to the time-varying random code:

,

Among them, c is a constant, δ ( x , y ) is a two-dimensional unit pulse function;

4) Time-varying decoding of the image captured by the CCD camera (as shown in Figure 5) is as follows:

,

In the formula, p ( x , y ) is the point spread function of the convex lens L ₂ 8;

5) Decode the coded images at all moments to obtain the super-resolution reconstructed image (as shown in Figure 4). Specifically, the reconstructed image of the time-varying decoded image o ₂ ( x , y , t ) is as follows :

,

In this example, the integral upper limit t is set to 1000. p (0 , 0) in Equation (4) is the value of the central point of the point spread function, and o ( x , y ) is the super-resolution reconstructed image of the original image s ( x , y ).

It can be seen from the comparative combination diagram in Figure 5 of the experimental results that the three concentric rings in the original image cannot be identified due to the lens point spread function; through the technical solution of this patent, the limitation of the diffraction limit can be broken through, and the blurred image can be reconstructed Construct the original image.

In this patent, digital images with nanometer resolution can be obtained through time-division multiplexing encoding of images by random nanoparticles, collection of wide-field images of traditional optical microscopes by CCD cameras, and decoding generated by computers based on random encoding.

The above description is only a preferred embodiment of the present invention, and of course it cannot be used to limit the scope of rights of the present invention. It should be pointed out that for those of ordinary skill in the art, any modification or equivalent replacement of the technical solutions of the present invention will Do not depart from the scope of protection of the technical solution of the present invention.

Claims (3)

1. A device for super-resolution microscopic imaging, characterized in that it comprises a blue LED light source (1), a convex lens L ₁ (2), a low-pass filter O1 (3), a dichroic prism (4), an encoder ( 5), high-pass filter O2 (7), convex lens L ₂ (8), CCD camera (9) and computer (10), the blue light emitted by the blue LED light source (1) passes through convex lens L ₁ (2), low-pass Optical filter O1 (3), beam splitting prism (4), and encoder (5) irradiate the measured sample (6), and the emitted light generated by the measured sample (6) passes through the encoder (5), beam splitting prism in sequence (4), high-pass filter O2 (7), convex lens L ₂ (8), and then sent to the computer (10) by the CCD camera (9). 2. The device for super-resolution microscopic imaging according to claim 1, characterized in that: the encoder (5) is a random nanoparticle encoder. 3. Utilize the method that the device of super-resolution microscopic imaging of claim 2 carries out microscopic imaging, it is characterized in that: comprise the following steps: 1), the blue light LED light source (1) emits blue light and is collimated by the convex lens L ₁ (2), the low-pass low-pass filter O1 (3), and the dichroic prism (4) excite the measured sample to generate emitted light s ( x , y ); 2), the encoder (5) generates a time-varying random code f ( x , y , t ) through nanoparticles, and encodes the emitted light s ( x , y ) of the sample to be measured, and passes through the spectroscopic prism (4), Qualcomm Qualcomm The optical filter O2 (7) and the convex lens L ₂ (8) focus on the image plane of the CCD camera (9); 3) The computer generates time-varying decoding f´ ( x , y , t ) according to the time-varying random code: , Among them, c is a constant, δ ( x , y ) is a two-dimensional unit pulse function; 4) Time-varying decoding of the images collected by the CCD camera is as follows: , In the formula, p ( x , y ) is the point spread function of the convex lens L ₂ (8); 5) Reconstruct the time-varying decoded image o ₂ ( x , y , t ) as follows: In the above formula, p (0 , 0) is the value of the center point of the point spread function, and o ( x , y ) is the super-resolution reconstructed image of the original image s ( x , y ).