阅读说明：本技术 多光谱显微自动聚焦装置及方法 (Multispectral microscopic automatic focusing device and method ) 是由 易定容 叶一青 孔令华 朱星星 蒋威 于 2019-11-21 设计创作，主要内容包括：本发明具体涉及一种多光谱显微自动聚焦装置及方法,该装置包括：复色光照明模块、纵向色散增强型光学成像模块、多光谱图像传感器,高精度载物台,图像分析及载物台控制系统,所述图像分析及载物台控制系统用于根据获得的离焦量值及离焦方向信号调节高精度载物台至光学成像系统的聚焦面,完成自动聚焦。该装置能实现一次成像即可快速获取样本离焦量及离焦方向,该方法能实现毫米量级大纵向测量范围的显微自动聚焦,相比传统显微自动聚焦方法,一方面,无需复杂的焦面搜索算法和昂贵的外部辅助设备,另一方面,提高了对焦效率和对焦精度。(The invention relates to a multispectral microscopic automatic focusing device and a method, wherein the device comprises: the device comprises a polychromatic light illumination module, a longitudinal dispersion enhanced optical imaging module, a multispectral image sensor, a high-precision objective table and an image analysis and objective table control system, wherein the image analysis and objective table control system is used for adjusting the high-precision objective table to a focusing surface of the optical imaging system according to the obtained defocusing value and defocusing direction signal to finish automatic focusing. The device can realize one-time imaging to quickly obtain the defocusing amount and the defocusing direction of the sample, and the method can realize microscopic automatic focusing in a millimeter-magnitude large longitudinal measurement range.)

1. A multispectral microscopic automatic focusing device is characterized by comprising a polychromatic light illumination module, a longitudinal dispersion enhanced optical imaging module and an image analysis and objective table control module,

the compound color light illumination module is sequentially provided with the following components in the light path propagation direction: a polychromatic light source (1), a condenser (2), a uniform collimating light lens group (3), a semi-reflecting and semi-transmitting spectroscope (4) and an objective lens (5) with longitudinal dispersion,

the longitudinal dispersion enhanced optical imaging module is sequentially provided with the following components in the light path propagation direction: the device comprises a high-precision objective table (6), an objective lens (5) with longitudinal dispersion, a semi-reflecting and semi-transmitting spectroscope (4), a tube mirror (7) with longitudinal dispersion and a multispectral image sensor (8);

the image analysis and objective table control module is sequentially provided with a multispectral image sensor (8), an image analysis and objective table control system (9) and a high-precision objective table (6) according to the transmission direction of a control signal;

the polychromatic light illumination module and the longitudinal dispersion enhanced optical imaging module share the semi-reflecting semi-transparent spectroscope (4) and the objective lens (5) with longitudinal dispersion;

the longitudinal dispersion enhanced optical imaging module and the image analysis and stage control module share the multispectral image sensor (8) and the high-precision stage (6);

the image analysis and stage control system (9) can be based on the sharpness F of each spectral gray level image_nAnd obtaining a defocusing value and a defocusing direction signal, and further adjusting the high-precision objective table (6) to a focusing surface of the optical imaging system to finish automatic focusing.

2. The multispectral microscopic auto-focusing device according to claim 1, wherein: the longitudinal dispersion enhanced optical imaging module comprises at least one objective lens (5) with longitudinal dispersion or at least one tube lens (7) with longitudinal dispersion.

3. The multispectral microscopic auto-focusing device according to claim 1, wherein: the longitudinal dispersion enhanced optical imaging module needs to eliminate transverse dispersion.

4. Multispectral microscopy according to claim 1An autofocus device, characterized by: the multispectral image sensor (8) can simultaneously acquire N sample gray level images I with different central wavelength spectral bands at zero time difference_nWherein N is more than or equal to 2, and N is more than or equal to 1 and less than or equal to N.

5. A multispectral microscopic auto-focusing method is characterized in that: use of the multispectral microscopic autofocus device of any of claims 1 to 4, comprising the steps of:

step 1, a sample to be detected is placed on a high-precision objective table (6);

step 2, adjusting the high-precision objective table (6) to enable the multispectral imaging sensor (8) to clearly image the sample in a certain imaging spectral band;

step 3, obtaining a sample gray level image I under N spectral bands with different central wavelengths through the multispectral image sensor (8)_n，1≤n≤N，N≥2；

Step 4, calculating the definition value F of each spectrum gray level image_nThe sharpness value F_nThe method can be used for calculating according to any one of the image definition evaluation functions of the Laplacian function, the Brenner function and the Tenengrad function;

step 5, the definition value F of the gray level image under two adjacent spectral bands with the maximum definition_nPerforming difference processing to obtain a defocus differential signal F_D＝F_n-1-F_n；

Step 6, through the out-of-focus differential signal F of the scale in advance_DCalculating and acquiring a defocusing amount value and a defocusing direction signal according to a defocusing amount relation curve;

and 7, adjusting the high-precision objective table (6) to the focal plane of the optical imaging system through the image analysis and objective table control system (9) according to the obtained defocus value and defocus direction signal, and finishing automatic focusing.

6. The method of multispectral microscopic auto-focusing according to claim 5, wherein: the method also comprises a method for judging the defocusing direction of the sample, and the specific judging process is as follows:

1) when a sample is focused, the spectral band with the maximum image definition value is taken as a central band;

2) if one of the two adjacent spectral bands with the maximum definition is a central band which is a longer band, and the definition value of the spectral image of the central band is greater than that of the spectral image of the other band, the point is on a positive defocusing surface of the optical imaging system and the defocusing amount is smaller;

3) if one of the two adjacent spectral bands with the maximum definition is a central band which is a longer band, and the definition value of the spectral image of the central band is smaller than that of the spectral image of the other band, the point is on a positive defocusing surface of the optical imaging system and the defocusing amount is larger;

4) if one of the two adjacent spectral bands with the maximum definition is a central band which is a shorter band, and the spectral image definition value of the central band is greater than that of the other band, the point is on a negative defocusing surface of the optical imaging system and the defocusing amount is smaller;

5) if one of the two adjacent spectral bands with the maximum definition is a central band which is a shorter band, and the spectral image definition value of the central band is smaller than that of the other band, the point is on a negative defocusing surface of the optical imaging system and the defocusing amount is larger;

6) if the two adjacent spectral bands with the maximum definition do not comprise the central band and the two bands are smaller than the central band, the point is on a positive defocusing surface of the optical imaging system, and the defocusing amount is large;

7) if the two adjacent spectral bands with the highest definition do not include the central band and the two bands are larger than the central band, the point is on the negative defocusing surface of the optical imaging system and the defocusing amount is large.

Technical Field

The invention belongs to the field of optical microscopic imaging, relates to a microscopic automatic focusing device and method, and particularly relates to a multispectral-based microscopic automatic focusing device and method.

Background

The existing microscopic automatic focusing technology has a plurality of defects. The passive microscopic automatic focusing technology directly performs focusing according to an imaging result, and a complex focal plane search algorithm and an image definition evaluation algorithm are required. The active microscopic automatic focusing technology needs to be combined with external auxiliary equipment to detect the defocusing amount and the defocusing direction, and the device is often complex in structure, expensive in price and needs a high-precision calibration process.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a multispectral microscopic automatic focusing device and a microscopic automatic focusing method. Compared with the traditional microscopic automatic focusing method, on one hand, the device does not need a complex focal plane search algorithm and expensive external auxiliary equipment, and on the other hand, the focusing efficiency and the focusing precision are improved.

The purpose of the invention is realized as follows:

the multispectral microscopic automatic focusing device comprises a polychromatic light illumination module, a longitudinal dispersion enhanced optical imaging module and an image analysis and objective table control module.

The above-mentioned compound color light illumination module has set gradually according to light path propagation direction: the device comprises a polychromatic light source, a condenser, a uniform collimating light lens group, a semi-reflecting and semi-transmitting spectroscope and an objective lens with longitudinal dispersion.

The longitudinal dispersion enhanced optical imaging module is sequentially provided with the following components in the light path propagation direction: the device comprises a high-precision objective table, an objective lens with longitudinal dispersion, a semi-reflecting semi-transmitting spectroscope, a tube lens with longitudinal dispersion and a multispectral image sensor.

The polychromatic light illumination module and the longitudinal dispersion enhanced optical imaging module share a semi-reflecting semi-permeable spectroscope and an objective lens with longitudinal dispersion.

The image analysis and objective table control module sequentially comprises the following components in the transmission direction of the control signal: multispectral image sensor, image analysis and objective table control system, high accuracy objective table.

The longitudinal dispersion enhanced optical imaging module and the image analysis and object stage control module share a multispectral image sensor and a high-precision object stage.

And the image analysis and objective table control system adjusts the high-precision objective table to the focal plane of the optical imaging system according to the obtained defocus value and defocus direction signal to finish automatic focusing.

The longitudinal dispersion enhanced optical imaging module comprises at least one objective lens with longitudinal dispersion, or comprises at least one tube lens with longitudinal dispersion, or other beneficial combinations.

The longitudinal dispersion enhanced optical imaging module has different focal lengths or image distances for optical signals of different wave bands, namely, under the same object distance, the object clear imaging axial positions of different wave bands are different, and the object surface height can be reversely deduced according to the clear imaging wave bands of the multispectral image sensor.

The longitudinal dispersion enhanced optical imaging module needs to eliminate transverse dispersion.

The multispectral image sensor can simultaneously acquire N (N is more than or equal to 2) sample gray level images I with different central wavelength spectral bands at zero time difference_n，1≤n≤N。

The multispectral microscopic automatic focusing method realized on the multispectral microscopic automatic focusing device comprises the following operation steps:

step 1, placing a sample to be detected on a high-precision objective table;

step 2, adjusting the high-precision objective table to enable a certain imaging spectral band of the multispectral imaging sensor to clearly image the sample;

step 3, acquiring N (N is more than or equal to 2) sample gray level images I under different central wavelength spectral bands through the multispectral image sensor_n，1≤n≤N；

Step 4, calculating the definition value F of each spectrum gray level image_nThe sharpness value F_nThe calculation can be carried out according to image definition evaluation functions such as a Laplacian function, a Brenner function, a Tenengrad function and the like;

step 5For the definition value F of the gray level image under two adjacent spectral bands with the maximum definition_nPerforming difference processing to obtain a defocus differential signal F_D＝F_n-1-F_n；

Step 6, through the out-of-focus differential signal F of the scale in advance_DCalculating and acquiring a defocusing amount value and a defocusing direction signal according to a defocusing amount relation curve;

and 7, adjusting the high-precision objective table to the focal plane of the optical imaging system by the image analysis and objective table control system according to the obtained defocus value and defocus direction signal to finish automatic focusing.

The multispectral microscopic automatic focusing method further comprises a method for judging the defocusing direction of the sample, and specifically comprises the following steps:

1) when a sample is focused, the spectral band with the maximum image definition value is taken as a central band;

2) if one of the two adjacent spectral bands with the maximum definition is a central band which is a longer band, and the definition value of the spectral image of the central band is greater than that of the spectral image of the other band, the point is on a positive defocusing surface of the optical imaging system and the defocusing amount is smaller;

3) if one of the two adjacent spectral bands with the maximum definition is a central band which is a longer band, and the definition value of the spectral image of the central band is smaller than that of the spectral image of the other band, the point is on a positive defocusing surface of the optical imaging system and the defocusing amount is larger;

4) if one of the two adjacent spectral bands with the maximum definition is a central band which is a shorter band, and the spectral image definition value of the central band is greater than that of the other band, the point is on a negative defocusing surface of the optical imaging system and the defocusing amount is smaller;

5) if one of the two adjacent spectral bands with the maximum definition is a central band which is a shorter band, and the spectral image definition value of the central band is smaller than that of the other band, the point is on a negative defocusing surface of the optical imaging system and the defocusing amount is larger;

6) if the two adjacent spectral bands with the maximum definition do not comprise the central band and the two bands are smaller than the central band, the point is on a positive defocusing surface of the optical imaging system, and the defocusing amount is large;

7) if the two adjacent spectral bands with the highest definition do not include the central band and the two bands are larger than the central band, the point is on the negative defocusing surface of the optical imaging system and the defocusing amount is large.

Compared with the existing microscopic automatic focusing technology, the method can realize the rapid acquisition of the defocusing amount and the defocusing direction of the sample by one-time imaging based on the multispectral dispersion principle, can realize the microscopic automatic focusing with millimeter magnitude and large longitudinal measurement range, and on one hand, does not need a complex focal plane search algorithm and expensive external auxiliary equipment, and on the other hand, improves the focusing efficiency and the focusing precision compared with the traditional microscopic automatic focusing method.

Drawings

Fig. 1 is a schematic diagram of the structure of the multispectral microscopic auto-focusing device.

In the figure: the system comprises a 1-polychromatic light source, a 2-condenser, a 3-uniform collimated light lens group, a 4-semi-reflecting and semi-transmitting spectroscope, a 5-objective lens with longitudinal dispersion, a 6-high-precision objective table, a 7-tube lens with longitudinal dispersion, an 8-multispectral image sensor and a 9-image analysis and objective table control system.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.