CN114112322A - Microscope focus offset measurement method based on differential confocal - Google Patents
Microscope focus offset measurement method based on differential confocal Download PDFInfo
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- CN114112322A CN114112322A CN202111225271.3A CN202111225271A CN114112322A CN 114112322 A CN114112322 A CN 114112322A CN 202111225271 A CN202111225271 A CN 202111225271A CN 114112322 A CN114112322 A CN 114112322A
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Abstract
The invention discloses a microscope focus offset measuring method based on differential confocal, which solves the problems of low focusing precision and low focusing speed of a microscope for samples to be measured with different thicknesses and focusing plane offset caused by switching different immersion objectives in the prior art, and comprises the following steps: s1: placing a sample to be tested; s2: imaging by a light source; s3: initializing a focusing position; s4: calculating difference; s5: adjusting the objective lens and the bias module; the reflected light of the sample to be measured is imaged through the three independent image sensors, the photometric distances from the three image sensors to the sample to be measured are inconsistent, the images of the three image sensors are axial response curves of light intensity changes, differential calculation is carried out on the formed images on the three image sensors, the defocusing amount of the objective lens with immersed media can be accurately calculated, accurate focusing is achieved, and accurate focusing on the sample to be measured with different thicknesses can be achieved through adjustment of the offset module.
Description
Technical Field
The invention relates to the technical field of automatic focusing of a human microscope, in particular to a microscope focus offset measurement method based on differential confocal.
Background
In long-time living cell observation experiments, due to the existence of factors such as temperature change, vibration, mechanical instability and the like, a focus offset phenomenon often occurs, and the focus offset phenomenon affects the definition of an observed image and even can cause the failure of the whole observation experiment. Therefore, the automatic focusing technology is produced. Autofocus techniques are classified into two types, active and passive. The common passive focusing method is based on an image processing technology, the position of a focal plane is determined by comparing the definition of images at different positions in front of and behind a plurality of focal planes, but the method has low focusing speed and poor real-time performance, and an accurate focusing position cannot be found when a biological sample with a certain thickness is focused. The existing active focusing method is to calculate the focus offset through the distance measured by external measuring equipment, the sensitivity and the precision of the method are lower, and the light spot obtained by the measuring equipment under the out-of-focus condition is very large and is difficult to determine the actual central position, so that a large error exists.
When observing a biological sample, it is necessary to use an objective lens of different magnification, and the objective lens is classified into a dry objective lens and an objective lens according to the immersion method characteristics of the objective lens. The existing active focusing system only considers the automatic focusing treatment of the dry lens, does not consider the characteristic that an additional layer of immersion medium exists in the objective lens, and has larger error during focusing. Due to the difference of the refractive indexes, the light of the detection light path is reflected before reaching the sample to be detected, and interferes with the light reflected by the sample to be detected, so that two light spots with close distances are formed on a detection sensor, and the system cannot distinguish a plane needing accurate focusing, so that the system cannot work normally.
Disclosure of Invention
The present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for measuring focus offset of a microscope based on differential confocal, which can achieve fast focusing and precise focusing of the microscope, and can solve the problem of focus plane offset caused by different immersion media on each objective when the objective is switched by the microscope.
In order to achieve the above object, the present invention provides a method for measuring focus offset of a microscope based on differential confocal, comprising the steps of:
s1: placing a sample to be tested: installing an objective lens with preset magnification on a microscope, wherein the objective lens is provided with an immersion medium and a sample to be detected is placed on one side of the objective lens;
s2: light source imaging: starting a light source module, irradiating illumination light rays emitted by the light source module onto a sample to be measured sequentially through a bias module and an objective lens, reflecting the illumination light rays by the sample to be measured, allowing the reflected light rays to enter an offset measurement module sequentially through the objective lens and the bias module, and respectively imaging on three image sensors in the offset measurement module;
s3: initializing a focusing position: superposing a focal plane of the objective lens and a plane where the reflected light of the microscope focal offset measurement module is located;
s4: and (3) difference calculation: collecting the formed images on the three image sensors, and carrying out differential calculation on the three images to measure the defocusing amount of the objective lens and the bias module;
s5: adjusting the objective lens and the bias module: and according to the measured defocusing amount of the objective lens and the bias module, driving the bias module to horizontally move for a preset distance to compensate the defocusing amount of the bias module, and driving the objective lens to vertically move for a preset height to compensate the defocusing amount of the objective lens.
According to the differential confocal microscope focus offset measurement method, the light source module comprises a system light source, a linear slit, a first reflector, a first collimating mirror and a semi-reflecting mirror, and illumination light emitted by the system light source sequentially passes through the linear slit, the first reflector, the first collimating mirror and the semi-reflecting mirror and is emitted into the offset module.
According to the differential confocal-based microscope focus offset measurement method, the system light source is an LED.
According to the differential confocal microscope focus offset measurement method, the bias module comprises a fixed lens, a movable lens and a driving piece, the fixed lens is arranged on one side of the semi-reflecting mirror, the movable lens is arranged on one side of the fixed lens close to the semi-reflecting mirror, and the movable lens can move towards one side far away from or close to the fixed lens through the driving piece.
According to the microscope focus offset measuring method based on differential confocal, the driving part is a stepping motor, and the stepping motor is connected with the movable lens.
According to the microscope focus offset measurement method based on differential confocal, the second reflector is arranged between the movable lens and the objective lens, and the irradiation light emitted by the system light source is irradiated on the sample to be measured through the fixed lens, the movable lens, the second reflector and the objective lens in sequence.
According to the microscope focus offset measurement method based on differential confocal, the offset measurement module comprises a first imaging light path, a second imaging light path and a third imaging light path, a first image sensor is arranged in the first imaging light path, a second image sensor is arranged in the second imaging light path, a third image sensor is arranged in the third imaging light path, and the first image sensor, the second image sensor and the third image sensor are all conjugated with the LED.
According to the above microscope focus offset measurement method based on differential confocal, the first imaging optical path includes a first cubic beam splitter, a first cylindrical lens and a first pinhole diaphragm, the second imaging optical path includes a second cubic beam splitter, a second cylindrical lens and a second pinhole diaphragm, the third imaging optical path includes a third cylindrical lens and a third pinhole diaphragm, the reflected light beam is divided into a first reflected light beam and a second reflected light beam by the first cubic beam splitter, the first reflected light beam is transmitted to the first image sensor through the first cylindrical lens and the first pinhole diaphragm in sequence, the second reflected light beam is divided into a third reflected light beam and a fourth reflected light beam through the second cubic beam splitter, the third reflected light beam is transmitted to the second image sensor through the second cylindrical lens and the second pinhole diaphragm in sequence, and the fourth reflected light beam is transmitted to the third image sensor through the third cylindrical lens and the third pinhole diaphragm in sequence On the sensor.
According to the differential confocal microscope focus offset measurement method, a first preset luminosity distance is set when the reflected light moves from the sample to be measured to the first image sensor, a second preset luminosity distance is set when the reflected light moves from the sample to be measured to the second image sensor, a third preset luminosity distance is set when the reflected light moves from the sample to be measured to the third image sensor, the second preset luminosity distance is smaller than the first preset luminosity distance, and the third preset luminosity distance is larger than the first preset luminosity distance.
According to the microscope focus offset measurement method based on differential confocal, a second collimating lens is arranged between the offset module and the offset measurement module.
The invention has the following beneficial effects:
1. because the first preset luminosity distance from the first image sensor to the sample to be measured is different from the second preset luminosity distance from the second image sensor to the sample to be measured and the third preset luminosity distance from the third image sensor to the sample to be measured, after reflected light is respectively formed on the three image sensors, imaging images on the three image sensors are all axial light intensity distribution curves, and the three images are subjected to differential operation to obtain the position of a light spot, namely an accurate focusing position;
2. for samples to be measured with different thicknesses, the conditions of different axial positions of the samples to be measured can be conveniently observed by adjusting the movable lens in the offset module, so that focusing is more accurate;
3. the pinhole diaphragm and the slit light source play a role in spatial filtering, stray light with other spatial frequencies is prevented from passing through, interference of reflected light spots on other surfaces of the sample is eliminated, and therefore imaging quality can be improved;
4. the system adopts a plurality of reflecting mirrors to change the path and the direction of the light beam, so that the overall layout is more compact.
Drawings
FIG. 1 is an overall process flow diagram of the present invention;
FIG. 2 is a schematic diagram of an optical delay line structure according to the present invention;
in the figure: 1. a system light source; 2. a linear slit; 3. a first reflector; 4. a first collimating mirror; 5. a half mirror; 6. a biasing module; 7. a second reflector; 8. an objective lens; 9. a sample to be tested; 10. a second collimating mirror; 11. an offset measurement module; 12. a first cubic beam splitter; 13. a first cylindrical lens; 14. a first pinhole diaphragm; 15. a first image sensor; 16. a second cubic beam splitter; 17. a third image sensor; 18. a second image sensor; 19. fixing the lens; 20. a movable lens; 21. a second cylindrical lens; 22. a second pinhole diaphragm; 23. a third cylindrical lens; 24. a third pinhole diaphragm; 25. a stepper motor.
Detailed Description
The following are specific embodiments of the present invention and are further described with reference to the drawings, but the present invention is not limited to these embodiments.
As shown in fig. 1-2, a method for measuring focus offset of a microscope based on differential confocal comprises the following steps:
s1: placing a sample to be tested 9: an objective lens 8 with a preset multiplying power is installed on a microscope, a sample 9 to be measured is placed on one side of the objective lens 8, an immersion medium is arranged on the objective lens 8, the medium can be air, water or oil, the medium can affect focusing accuracy during focusing, the refractive indexes of different immersion media are different, therefore, certain influence is caused on the focusing accuracy, and the multiplying power of the objective lens 8 is selected according to the actual requirements of an operator.
S2: light source imaging: the method comprises the steps that a light source module is started, illumination light emitted by the light source module sequentially passes through a bias module 6 and an objective lens 8 to irradiate a sample 9 to be measured, the illumination light sequentially passes through the objective lens 8 and enters an offset measurement module 11 through the bias module 6 and is imaged on three image sensors in the offset measurement module 11 respectively, then the light source module emits light, the reflected light is expanded through the bias module 6 and then irradiates the sample 9 to be measured through the objective lens 8, reflected light is generated on the sample 9 to be measured, the reflected light enters the offset measurement module 11 through the objective lens 8 and the bias module 6 and is imaged in the offset measurement module 11, due to the fact that the three image sensors are adopted, obtained light spots are small and energy is concentrated, operation aiming at light spot focal coordinates is not required to be additionally added, and the operation amount of subsequent data processing is reduced.
S3: initializing a focusing position: the focal plane of the objective lens 8 is overlapped with the plane where the reflected light of the microscope focal offset measuring module 11 is located, the position of the light reflection plane can be correspondingly changed according to different immersion media used by the objective lens 8, and the focal plane of the objective lens 8 needs to be manually adjusted to be overlapped with the plane where the reflected light of the microscope focal offset measuring module 11 is located, so that the initial focusing position is ensured, and the objective lens 8 is suitable for different immersion media.
S4: and (3) difference calculation: the forming images on the three image sensors are collected, the three images are subjected to differential calculation to measure the defocusing amount of the objective lens 8 and the offset module 6, the imaging conditions of the three image sensors are inconsistent, the three obtained forming images are subjected to intensity differential calculation to obtain a final higher-resolution microscopic image, and the defocusing amount of the objective lens 8 and the offset module 6 is measured by the higher-resolution microscopic image.
S5: adjusting the objective lens 8 and the bias module 6: according to the measured defocusing amount of the objective lens 8 and the offset module 6, the offset module 6 is driven to horizontally move for a preset distance to compensate the defocusing amount of the offset module 6, the objective lens 8 is driven to vertically move for a preset height to compensate the defocusing amount of the objective lens 8, the vertical height of the objective lens 8 is adjusted through the microscope shelf to compensate the defocusing amount of the objective lens 8 according to the measured defocusing amount, and the defocusing amount of the offset module 6 is compensated through the horizontal movement of the offset module 6, so that accurate focusing is realized.
In the present embodiment, three image sensors are used to obtain a higher-resolution microscopic image, so as to achieve precise focusing, and when focusing accuracy is not required, one image sensor may be used to achieve rapid focusing.
The light source module comprises a system light source 1, a linear slit 2, a first reflector 3, a first collimating mirror 4 and a semi-reflecting mirror 5, illumination light rays emitted by the system light source 1 sequentially pass through the linear slit 2, the first reflector 3, the first collimating mirror 4 and the semi-reflecting mirror 5 to be emitted into the bias module 6, and the linear slit 2 can limit stray light and scattered light, so that after passing through the first reflector 3 and the first collimating mirror 4, the illumination light rays are changed into parallel light and enter the bias module 6 to be expanded.
The system light source 1 is an LED, and the LED emits illumination light.
The driving member is a stepping motor 25, the stepping motor 25 is connected with the movable lens 20, and the stepping motor 25 provides power for the movement of the movable lens 20.
The movable lens 20 and the objective lens 8 are provided with the second reflector 7 therebetween, the irradiation light emitted by the system light source 1 irradiates on the sample 9 to be measured through the fixed lens 19, the movable lens 20, the second reflector 7 and the objective lens 8 in sequence, and the light irradiates on the sample 9 to be measured after converging through the objective lens 8 after being expanded through the bias module 6.
The offset measurement module 11 comprises a first imaging light path, a second imaging light path and a third imaging light path, wherein a first image sensor 15 is arranged in the first imaging light path, a second image sensor 18 is arranged in the second imaging light path, a third image sensor 17 is arranged in the third imaging light path, the first image sensor 15, the second image sensor 18 and the third image sensor 17 are all conjugated with the LED, and imaging images on the three image sensors are all axial light intensity distribution curves, so that difference calculation is facilitated.
The first imaging optical path comprises a first cubic beam splitter 12, a first cylindrical lens 13 and a first pinhole diaphragm 14, the second imaging optical path comprises a second cubic beam splitter 16, a second cylindrical lens 21 and a second pinhole diaphragm 22, the third imaging optical path comprises a third cylindrical lens 23 and a third pinhole diaphragm 24, the reflected light is divided into a first reflected light and a second reflected light by the first cubic beam splitter 12, the first reflected light is transmitted to the first image sensor 15 by the first cylindrical lens 13 and the first pinhole diaphragm 14 in sequence, the second reflected light is divided into a third reflected light and a fourth reflected light by the second cubic beam splitter 16, the third reflected light is transmitted to the second image sensor 18 by the second cylindrical lens 21 and the second pinhole diaphragm 22 in sequence, the fourth reflected light is transmitted to the third image sensor 17 by the third cylindrical lens 23 and the third pinhole diaphragm 24 in sequence, the reflected light is divided into three reflected lights by the first cubic beam splitter 12 and the second cubic beam splitter 16, and the three reflected lights are individually formed on three image sensors, and the three image sensors are all provided with a data processing unit and a data storage unit.
The reflected light moves from the sample to be measured to said first image sensor 15 a first predetermined photometric distance, the reflected light moves from the sample to be measured to a second predetermined photometric distance from the second image sensor 18, the reflected light moves from the sample to be measured to a third predetermined photometric distance of the third image sensor 17, the second preset photometric distance is less than the first preset photometric distance, the third preset photometric distance is greater than the first preset photometric distance, in this embodiment, the first predetermined luminous distance is X, the second predetermined luminous distance is Y, the third predetermined luminous distance is Z, X ═ Y + d, X ═ Z-d, that is, the difference between the first preset photometric distance and the preset photometric distance is equal to the difference between the third preset photometric distance and the first preset photometric distance, so as to obtain an axial light intensity distribution curve.
A second collimating mirror 10 is arranged between the bias module 6 and the deviation measuring module 11, and is used for converting the reflected light rays into parallel light.
Aiming at an objective lens 8, reflected light of a sample 9 to be measured is imaged through three independent image sensors, the photometric distances from the three image sensors to the sample to be measured are different, the images of the three image sensors are all axial response curves of light intensity change, the defocusing amount of the objective lens 8 with immersed media can be accurately calculated through differential calculation of formed images on the three image sensors, accurate focusing is achieved, and accurate focusing of samples to be measured with different thicknesses can be achieved through adjustment of a bias module 6.
The technical solutions of the present invention are explained in detail above with reference to the accompanying drawings, and the described embodiments are used to help understanding the idea of the present invention. The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (10)
1. A microscope focus offset measurement method based on differential confocal is characterized by comprising the following steps:
s1: placing a sample to be tested: installing an objective lens with preset magnification on a microscope, arranging an immersion medium on the objective lens, and placing a sample to be detected on one side of the objective lens;
s2: light source imaging: starting a light source module, irradiating illumination light rays emitted by the light source module onto a sample to be measured sequentially through a bias module and an objective lens, reflecting the illumination light rays by the sample to be measured, allowing the reflected light rays to enter an offset measurement module sequentially through the objective lens and the bias module, and respectively imaging on three image sensors in the offset measurement module;
s3: initializing a focusing position: superposing a focal plane of the objective lens and a plane where the reflected light of the microscope focal offset measurement module is located;
s4: and (3) difference calculation: collecting the formed images on the three image sensors, and carrying out differential calculation on the three images to measure the defocusing amount of the objective lens and the bias module;
s5: adjusting the objective lens and the bias module: and according to the measured defocusing amount of the objective lens and the bias module, driving the bias module to horizontally move for a preset distance to compensate the defocusing amount of the bias module, and driving the objective lens to vertically move for a preset height to compensate the defocusing amount of the objective lens.
2. The method as claimed in claim 1, wherein the light source module includes a system light source, a linear slit, a first reflector, a first collimating mirror and a half-reflector, and the illumination light emitted from the system light source is sequentially emitted into the deflection module through the linear slit, the first reflector, the first collimating mirror and the half-reflector.
3. The differential confocal-based microscope focus offset measurement method according to claim 2, wherein the system light source is an LED.
4. The differential confocal-based microscope focus offset measurement method according to claim 2, wherein the bias module comprises a fixed lens, a movable lens and a driving member, the fixed lens is disposed on one side of the half mirror, the movable lens is disposed on one side of the fixed lens close to the half mirror, and the movable lens can move towards one side far away from or close to the fixed lens through the driving member.
5. The differential confocal-based microscope focus offset measurement method according to claim 4, wherein the driving member is a stepping motor, and the stepping motor is connected to the movable lens.
6. The method as claimed in claim 4, wherein a second reflector is disposed between the movable lens and the objective lens, and the illumination light from the system light source is sequentially emitted onto the sample to be measured through the fixed lens, the movable lens, the second reflector and the objective lens.
7. The method as claimed in claim 3, wherein the shift measurement module includes a first imaging optical path, a second imaging optical path, and a third imaging optical path, the first imaging optical path has a first image sensor disposed therein, the second imaging optical path has a second image sensor disposed therein, the third imaging optical path has a third image sensor disposed therein, and the first image sensor, the second image sensor, and the third image sensor are all conjugated to the LED.
8. The method of claim 7, wherein the first imaging optical path comprises a first cube beam splitter, a first cylindrical lens and a first pinhole diaphragm, the second imaging optical path comprises a second cube beam splitter, a second cylindrical lens and a second pinhole diaphragm, the third imaging optical path comprises a third cylindrical lens and a third pinhole diaphragm, the reflected light beam is divided into a first reflected light beam and a second reflected light beam by the first cube beam splitter, the first reflected light beam is transmitted to the first image sensor through the first cylindrical lens and the first pinhole diaphragm, the second reflected light beam is divided into a third reflected light beam and a fourth reflected light beam through the second cube beam splitter, the third reflected light beam is transmitted to the second image sensor through the lens and the second pinhole diaphragm, the fourth reflected light ray passes through a third cylindrical lens and a third pinhole diaphragm in sequence and is transmitted to the third image sensor.
9. The method as claimed in claim 8, wherein a first predetermined photometric distance is provided for the reflected light moving from the sample to be measured to the first image sensor, a second predetermined photometric distance is provided for the reflected light moving from the sample to be measured to the second image sensor, a third predetermined photometric distance is provided for the reflected light moving from the sample to be measured to the third image sensor, the second predetermined photometric distance is smaller than the first predetermined photometric distance, and the third predetermined photometric distance is larger than the first predetermined photometric distance.
10. The method for measuring the focus offset of the microscope based on the differential confocal method according to claim 1 or 8, wherein a second collimating lens is arranged between the offset module and the offset measuring module.
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