A method for measuring the thickness of a fluorescent staining film by coupling interference and fluorescence in-situ

Abstract

The present invention provides a method for measuring the thickness of a fluorescently stained thin film by in-situ coupling of interference and fluorescence in-situ. Aiming at the problems that the interference fringe method is not applicable to the measurement of film thickness above the micron scale and the fluorescence method has poor sensitivity in measuring film thickness below the micron scale, the present invention takes into account the respective advantages of the two methods and uses the two methods based on the same optical path to measure the thickness of the thin film at the same position, realizing the mutual coupling of the fluorescence method and the optical interference method in-situ. That is, the calibration relationship of the fluorescence method can be corrected by the film thickness calculated by the optical interference method, so as to improve the sensitivity of the fluorescence method in measuring the film thickness below the micron scale and expand the effective range of the fluorescence method.

Description

Technical Field

[0001] The present invention relates to the field of measurement technology, and in particular to a method for measuring the thickness of a fluorescently stained thin film by in-situ coupling of interference and fluorescence. Background Art

[0002] The optical fluorescently stained thin film thickness measurement technology is a technology for measuring the thickness of a transparent fluorescently stained liquid or solid thin film based on an optical method, such as measuring the thickness of an LED fluorescent thin film, the thickness of a fluorescent liquid thin film in a gap, or indirectly measuring the height of a structural gap, the depth of a surface groove, etc., and is applied in industries such as machinery, electronics, and biology. The fluorescence method is a method for measuring the thickness of a transparent thin film based on the principle of laser-induced fluorescence. This method uses a laser to excite a fluorescently stained thin film with a standard thickness to obtain a fluorescence image, and through calculation and fitting, a calibration relationship between the thin film thickness and the fluorescence intensity is obtained; then, the thin film to be measured is uniformly stained with a fluorescent agent, a fluorescence image is obtained by laser excitation, and the thickness of the thin film corresponding to each pixel is calculated using the gray value of the fluorescence image.

[0003] However, the fluorescence method is generally used for measuring the thickness of thin films in the micron to millimeter scale, and the sensitivity for measuring the film thickness below the micron scale is poor. The optical interference method is based on the principle of laser interference to obtain a laser interference image of the thin film, and the gap thickness is calculated through the interference fringe order and the relative light intensity, and has high sensitivity in measuring the thickness of thin films at the nanometer scale. Therefore, if the fluorescence method and the optical interference method can be coupled with each other in-situ, the calibration relationship of the fluorescence method can be corrected by the thin film thickness calculated by the optical interference method, thereby improving the sensitivity of the fluorescence method in measuring the thickness of thin films below the micron scale and expanding the effective range of the fluorescence method. Summary of the Invention

[0004] In view of this, the present invention provides a method for measuring the thickness of a fluorescently stained thin film by in-situ coupling of interference and fluorescence, which can couple the fluorescence method and the optical interference method in-situ, and correct the calibration relationship of the fluorescence method by the thin film thickness calculated by the optical interference method, thereby improving the sensitivity of the fluorescence method in measuring the thickness of thin films below the micron scale and expanding the effective range of the fluorescence method.

[0005] A method for measuring the thickness of a fluorescently stained thin film by in-situ coupling of interference and fluorescence, the measurement system used includes an in-situ coupling main optical path of interference and fluorescence and a combination module of the thin film to be measured;

[0006] The combination module of the thin film to be measured includes a gold coating (3), a glass sheet (4), a micro-groove device (1), and a fluorescent staining medium (2); the lower surface of the glass sheet (4) is coated with a gold coating (3); the upper surface of the micro-groove device (1) is polished and etched with rectangular micro-grooves, and the bottom surface of the groove is used as a reflection surface; the micro-grooves are filled with a fluorescent staining medium (2);

[0007] The fluorescence emission peak wavelength of the fluorescence staining medium (2) is λ E ;

[0008] The interference-fluorescence in-situ coupling main optical path includes an objective lens (5), a first imaging lens (13), a second imaging lens (15), a fluorescence module, an optical interference module, a beam splitting module, a first camera (14), a second camera (16), a first laser source (7), and a second laser source (10);

[0009] The thin film combination module to be measured, the objective lens (5), the fluorescence module, the interference module, and the beam splitting module are arranged in sequence in the main optical path;

[0010] The fluorescence module includes a dichroic long-pass filter (17) with a threshold of λ1 and a first long-pass filter (18) with a threshold of λ2; the dichroic long-pass filter (17) is at a 45-degree angle to the optical axis of the main optical path, and the first long-pass filter (18) is placed perpendicular to the optical axis of the main optical path;

[0011] The optical interference module includes a full-wavelength semi-reflective and semi-transmissive filter (19); the full-wavelength semi-reflective and semi-transmissive filter (19) is at a 45-degree angle to the optical axis of the main optical path;

[0012] The beam splitting module includes a dichroic short-pass filter (20) with a threshold of λ3, a short-pass filter (21) with a threshold of λ4, and a second long-pass filter (22) with a threshold of λ5; the dichroic short-pass filter (20) is at a 45-degree angle to the optical axis of the main optical path; the short-pass filter (21) is perpendicular to the optical axis of the main optical path, and the second long-pass filter (22) is parallel to the optical axis of the main optical path;

[0013] The fluorescence module, the optical interference module, and the beam splitting module can be respectively moved into and out of the main optical path;

[0014] The first camera (14) and the first imaging lens (13) are placed in the reflection optical path of the second long-pass filter (22) for interference imaging;

[0015] The second camera (16) and the second imaging lens (15) are placed in the reflection optical path of the full-wavelength semi-reflective and semi-transmissive filter (19) for fluorescence imaging;

[0016] The working wavelength of the first laser source (7) is λ F , and the laser generated by it is reflected by the dichroic long-pass filter (17) and enters the main optical path; the working wavelength of the second laser source (10) is λ R , and it is reflected by the full-wavelength semi-reflective and semi-transmissive filter (19) and enters the main optical path;

[0017] The wavelength relationship satisfies the following formula:

[0018] λF <λ1 < λ2 < λ E <530nm < λ4 < λ3 < λ5 < λ R ;

[0019] The measurement method includes:

[0020] Step S11: Prepare K c micro-groove devices (1) with different groove depths, fill the micro-grooves with a fluorescent staining medium (2), and attach the surface of the gold coating (3) on the glass slide (4) to the top surface of the groove to form a standard fixed-thickness fluorescent staining film;

[0021] Step S12: Turn on the second laser source (10) and the second camera (16), focus on and photograph the standard fixed-thickness fluorescent staining film to obtain K c fluorescent images;

[0022] Step S13: Prepare K w micro-groove devices (1) with different micro-groove depths, contact any edge of the surface of the gold coating (3) on the glass slide (4) with the bottom surface of the groove, and use the fluorescent staining medium (2) to fill the wedge-shaped gap formed between the glass slide (4) and the micro-groove to form a standard wedge-shaped fluorescent staining film;

[0023] Step S14: Turn on the first laser source (7), the second laser source (10), the first camera (14), and the second camera (16), focus on the standard wedge-shaped fluorescent staining film, and photograph the fluorescent image and the interference fringe image;

[0024] Step S15: Repeat Step S14 for all standard wedge-shaped fluorescent staining films to obtain K w fluorescent images and interference fringe images;

[0025] Step S16: Use the optical interference method to calculate the film thickness of any pixel (i, j) in the k-th interference fringe image

[0026] where is the depth of the micro-groove at (i, jc); is the gray value at the edge of the micro-groove; is the number of extreme points at the edge of the micro-groove; r f is the refractive index of the fluorescent staining medium (2); jc is the pixel column coordinate of the position with the maximum thickness of the standard wedge-shaped fluorescent staining film of the micro-groove device (1), p i,j (k) is the number of gray extreme points between the pixel (i, j) and the pixel (i, jc) in the i-th row of pixels; where p i,jc(k) Indicates the number of extreme points at the groove edge, which is 0, g i,j (k) Is the gray value of pixel (i, j), g i,j (k)max Is the nearest gray maximum value to pixel (i, j) among the pixels in the i-th row, g i,j (k)min Is the nearest gray minimum value to pixel (i, j) among the pixels in the i-th row;

[0027] Step S17: Repeat S16 for K w Interference fringe images to obtain the film thickness corresponding to all interference fringe images;

[0028] Step S18: For K = K c + K w Fluorescence images to construct a calibration coefficient linear equation system:

[0029]

[0030] Where, a i,j (n) Is the coefficient of the n-th order fitting term corresponding to pixel (i, j), N is the total order of the fitting formula, h i,j (k) Is the film thickness corresponding to pixel (i, j) in the k-th image, where, K = K c + K w Among the K c The first K w Images are of a standard fixed-thickness fluorescently stained film with a known thickness, and the last K

[0031] Images are of a standard wedge-shaped fluorescently stained film, and the thickness is calculated according to the formula in S16;

[0032] Step S19: Solve the calibration coefficient linear equation system constructed in step S18 to obtain a fitting term coefficient table;

[0033] Step S20: Use the same optical measurement system to take a fluorescence photo of the film to be measured;

[0034]

[0035] Where, h i,j Is the thickness of the film to be measured at pixel (i, j), g i,j Is the gray value corresponding to pixel (i, j) of the fluorescence image of the film to be measured.

[0036] Preferably, λ1 = 490 nm; λ2 = 500 nm; λ3 = 625 nm; λ4 = 600 nm; λ5 = 630 nm; λ F = 470 nm; λ R = 655 nm; λ E = 515 nm.

[0037] Preferably, the first camera (14) and the second camera (16) are interconnected through a synchronization signal line (23) to achieve synchronous triggering.

[0038] Preferably, the fluorescence module further includes a first mirror mount (6); the dichroic long-pass filter (17) and the first long-pass filter (18) are mounted on the first mirror mount (6).

[0039] Preferably, the optical interference module further includes a second mirror mount (9); the full-wavelength semi-transparent and semi-reflective filter (19) is mounted on the second mirror mount (9).

[0040] Preferably, the beam splitting module further includes a third mirror mount (12); the dichroic short-pass filter (20), the short-pass filter (21) and the second long-pass filter (22) are mounted on the third mirror (12).

[0041] The present invention has the following beneficial effects:

[0042] The present invention provides a method for measuring the thickness of a fluorescently stained thin film by in-situ coupling of interference and fluorescence. Aiming at the problems that the interference fringe method is not applicable to the measurement of film thickness above the micron scale and the fluorescence method has poor sensitivity in measuring film thickness below the micron scale, the present invention takes into account the respective advantages of the two methods and uses the two methods based on the same optical path to measure the thickness of the thin film at the same position, realizing the mutual coupling of the fluorescence method and the optical interference method in-situ. Thus, the calibration relationship formula of the fluorescence method can be corrected by the film thickness calculated by the optical interference method, thereby improving the sensitivity of the fluorescence method in measuring the film thickness below the micron scale and expanding the effective range of the fluorescence method. Description of the Drawings

[0043] Figure 1 Schematic diagrams of the hardware system composition, imaging principle and actual usage mode of the measurement method of the present invention;

[0044] Figure 2 Diagram showing the corresponding relationship between the intensity curve, interference pattern and standard wedge-shaped fluorescently stained thin film of the interference image obtained by the measurement method of the present invention.

[0045] Wherein, 1 - mirror, 2 - fluorescently stained thin film to be measured, 3 - gold coating, 4 - glass sheet, 5 - objective lens, 6 - first mirror mount (fluorescence module), 7 - first laser source (wavelength λ Fnm, for interference imaging), 8 - first optical fiber, 9 - second mirror mount (interference module), 10 - second laser source (wavelength λ R nm, for fluorescence imaging), 11 - second optical fiber, 12 - third mirror mount (beam splitting module), 13 - first imaging lens (interference), 14 - first camera (interference), 15 - second imaging lens (fluorescence), 16 - second camera (fluorescence), 17 - dichroic long - pass filter (threshold λ1 nm), 18 - first long - pass filter (threshold λ2 nm), 19 - full - wavelength semi - reflecting and semi - transmitting filter, 20 - dichroic short - pass filter (threshold λ3 nm), 21 - short - pass filter (threshold λ4 nm), 22 - second long - pass filter (threshold λ5 nm), 23 - camera synchronization signal line. The arrowed dashed lines represent light rays and their propagation directions. Detailed implementation mode

[0046] The present invention will be described in detail below with reference to the accompanying drawings and by way of examples.

[0047] The present invention provides a method for measuring the thickness of a fluorescent - stained thin film by in - situ coupling of interference and fluorescence, based on Figure 1 the measurement system shown, including: an in - situ coupling main optical path of interference - fluorescence and a thin - film - to - be - measured combination module;

[0048] The thin - film - to - be - measured combination module includes a gold coating 3, a glass sheet 4, a micro - trench device 1, and a fluorescent - staining medium 2; the material of the glass sheet 4 is quartz glass, and a gold coating 3 is plated on its lower surface. The material of the gold coating 3 is high - purity gold, with silicon dioxide as a protective layer, and the surface is flat.

[0049] The micro - trench device 1 is made of single - crystal silicon or metal. Its upper surface is polished and etched with rectangular micro - trenches, and the bottom surface of the trenches has a high reflectivity; the micro - trenches are filled with the fluorescent - staining medium 2.

[0050] The fluorescent - staining medium 2 is a liquid or soft substance with high transparency and uniform fluorescent staining, and its fluorescence emission peak wavelength is λ E = 515 nm;

[0051] The glass sheet 4 is placed on the upper surface of the micro - trench device 1, with the gold coating 3 facing downwards.

[0052] The in - situ coupling main optical path of interference - fluorescence includes: an objective lens 5, a first imaging lens 13, a second imaging lens 15, a fluorescence module, an optical interference module, a beam splitting module, a first camera 14, a second camera 16, a first laser source 7, a second laser source 10, a first optical fiber 8, a second optical fiber 11, and a camera synchronization signal line 23;

[0053] The thin - film - to - be - measured combination module, the objective lens 5, the fluorescence module, the interference module, and the beam splitting module are arranged in sequence from bottom to top in the main optical path.

[0054] The fluorescence module includes: a dichroic long-pass filter 17 with a threshold of λ1 = 490 nm, a first long-pass filter 18 with a threshold of λ2 = 500 nm, and a first mirror mount 6; the dichroic long-pass filter 17 and the first long-pass filter 18 are mounted on the first mirror mount 6; the dichroic long-pass filter 17 forms an angle of 45 degrees with the optical axis of the main optical path, and the first long-pass filter 18 is placed perpendicular to the optical axis of the main optical path.

[0055] The optical interference module includes: a full-wavelength semi-reflective and semi-transmissive filter 19, and a second mirror mount 9, the full-wavelength semi-reflective and semi-transmissive filter 19; the full-wavelength semi-reflective and semi-transmissive filter 19 is mounted on the second mirror mount 9, making it form an angle of 45 degrees with the optical axis of the main optical path.

[0056] The spectroscopic module includes: a dichroic short-pass filter 20 with a threshold of λ3 = 625 nm, a short-pass filter 21 with a threshold of λ4 = 600 nm, a second long-pass filter 22 with a threshold of λ5 = 630 nm, and a third mirror mount 12. The dichroic short-pass filter 20, the short-pass filter 21, and the second long-pass filter 22 are mounted on the third mirror mount 12; the dichroic short-pass filter 20 forms an angle of 45 degrees with the optical axis of the main optical path; the short-pass filter 21 is perpendicular to the optical axis of the main optical path, and the second long-pass filter 22 is parallel to the optical axis of the main optical path.

[0057] The first mirror mount 6, the second mirror mount 9, and the third mirror mount 12 can all move parallelly, and the fluorescence module, the optical interference module, and the spectroscopic module can be respectively moved into and out of the main optical path to form different working modes according to needs.

[0058] The first camera 14 and the second camera 16 are two cameras with the same frame rate and resolution, and can be connected to each other using a synchronization signal line 23 to achieve synchronous triggering.

[0059] The first camera 14 and the first imaging lens 13 are placed in the reflection optical path of the second long-pass filter 22 for interference imaging.

[0060] The second camera 16 and the second imaging lens 15 are placed in the reflection optical path of the full-wavelength semi-reflective and semi-transmissive filter 19 for fluorescence imaging.

[0061] The working wavelength of the first laser source 7 is λ F = 470 nm and is used for fluorescence imaging. The laser generated by it is reflected by the dichroic long-pass filter 17 and enters the main optical path; the working wavelength of the second laser source 10 is λ R = 655 nm and is used for interference imaging. It is reflected by the full-wavelength semi-reflective and semi-transmissive filter 19 and enters the main optical path.

[0062] The laser sources and each filter satisfy the following wavelength relationship:

[0063] λ F <λ1 < λ2 < λ E <530nm < λ4 < λ3 < λ5 < λ R 。

[0064] For the described measurement method, several in-situ fluorescence images and interference fringe images of a standard thickness fluorescently stained film need to be obtained. The specific steps are as follows:

[0065] Step S11: Prepare K c micro-groove devices 1 with different groove depths. Fill the micro-groove devices 1 with a fluorescent staining medium 2, and closely attach the surface of the glass film 3 to the top surface of the groove to form a standard fixed-thickness fluorescently stained film; where K c is greater than 2, and the more the quantity, the better.

[0066] Step S12: Turn on the second laser source 10 and the second camera 16, focus on and photograph the standard fixed-thickness fluorescently stained film to obtain K c fluorescence images;

[0067] Step S13: Prepare K w micro-groove devices 1 with different groove depths. Contact any edge of the surface of the glass film 3 with the bottom surface of the groove, and fill the two wedge-shaped gaps formed between the glass sheet and the standard micro-groove with a fluorescent staining medium to form a standard wedge-shaped fluorescently stained film; K c and K w are greater than 2, and the more the quantity, the better.

[0068] Step S14: Turn on the first laser source 7, the second laser source 10, the first camera 14, and the second camera 16, focus on the standard wedge-shaped fluorescently stained film, adjust the orientation of the standard wedge-shaped fluorescently stained film so that the interference fringes are as parallel as possible to the column direction of the pixels, and the pixel column coordinate j increases as the wedge tip moves upward, and photograph the fluorescence image and the interference fringe image;

[0069] Step S15: Repeat Step S14 for all standard wedge-shaped fluorescently stained films to obtain K w in-situ fluorescence images and interference fringe images;

[0070] Step S16: For any pixel (i, j) of the k-th interference fringe image, calculate the film thickness using the optical interference method as follows:

[0071]

[0072] where, is the depth of the micro-groove at (i, jc); is the gray value at the edge of the micro-groove; is the number of extreme points at the edge of the micro-groove; rf is the refractive index of the fluorescence staining medium (2); jc is the pixel column coordinate of the maximum position of the standard wedge-shaped fluorescence staining film thickness of the microgroove device (1), p i,j (k) is the number of gray extreme points between the pixel (i, j) and the pixel (i, jc) in the i-th row of pixels; where p i,jc (k) represents the number of extreme points at the groove edge, which is 0, g i,j (k) is the gray value of the pixel (i, j), g i,j (k)max is the nearest gray maximum value to the pixel (i, j) in the i-th row of pixels, g i,j (k)min is the nearest gray minimum value to the pixel (i, j) in the i-th row of pixels;

[0073] Step S17: Repeat S16 for K w interference fringe images to obtain the film thickness corresponding to all interference fringe images;

[0074] Step S18: For K = K c + K w fluorescence images, construct a calibration coefficient linear equation system:

[0075]

[0076] where a i,j (n) is the coefficient of the n-th order fitting term corresponding to the pixel (i, j), N is the total order of the fitting formula, h i,j (k) is the film thickness corresponding to the pixel (i, j) in the k-th image, where K = K c + K w Among the K c fluorescence images, the first K w images are taken of a standard fixed-thickness fluorescence staining film with a known thickness, and the last K

[0077] Step S19: Solve the calibration coefficient linear equation system constructed in Step S18 to obtain a fitting term coefficient table;

[0078] Step S20: Use the same optical measurement system to take a fluorescence photo of the film to be measured;

[0079] Step S21: Use the fitting term coefficients calculated in Step S19 to calculate the film thickness corresponding to each pixel (i, j) according to the relationship between the film thickness and the fluorescence intensity value:

[0080]

[0081] Among them, h i,j is the thickness of the film to be measured at pixel (i, j), and g i,j is the gray value corresponding to the pixel (i, j) of the fluorescence image of the film to be measured.

[0082] In summary, the above are only the preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An interference-fluorescence in-situ coupled fluorescence staining film thickness measurement method, characterized in that The measurement system adopted includes an interference-fluorescence in-situ coupling main optical path and a thin film combination module to be measured; The thin film combination module to be measured includes a gold coating (3), a glass slide (4), a micro-groove device (1), and a fluorescence staining medium (2); the lower surface of the glass slide (4) is coated with a gold coating (3); the upper surface of the micro-groove device (1) is polished and etched with rectangular micro-grooves, and the bottom surface of the grooves serves as a reflecting surface; The fluorescence staining medium (2) is inside the micro-grooves; The fluorescence emission peak wavelength of the fluorescent staining medium (2) is λ E ; The interference-fluorescence in-situ coupling main optical path includes an objective lens (5), a first imaging lens (13), a second imaging lens (15), a fluorescence module, an optical interference module, a beam splitting module, a first camera (14), a second camera (16), a first laser source (7), and a second laser source (10); The thin film combination module to be measured, the objective lens (5), the fluorescence module, the interference module, and the beam splitting module are arranged in sequence in the main optical path; The fluorescence module includes a dichroic long-pass filter (17) with a threshold of λ1 and a first long-pass filter (18) with a threshold of λ2; the dichroic long-pass filter (17) is at an angle of 45 degrees with the optical axis of the main optical path, and the first long-pass filter (18) is placed perpendicular to the optical axis of the main optical path; The optical interference module includes a full-wavelength semi-reflective and semi-transmissive filter (19); the full-wavelength semi-reflective and semi-transmissive filter (19) is at an angle of 45 degrees with the optical axis of the main optical path; The beam splitting module includes a dichroic short-pass filter (20) with a threshold of λ3, a short-pass filter (21) with a threshold of λ4, and a second long-pass filter (22) with a threshold of λ5; the dichroic short-pass filter (20) is at an angle of 45 degrees with the optical axis of the main optical path; the short-pass filter (21) is perpendicular to the optical axis of the main optical path, and the second long-pass filter (22) is parallel to the optical axis of the main optical path; The fluorescence module, the optical interference module, and the beam splitting module can be respectively moved into and out of the main optical path; The first camera (14) and the first imaging lens (13) are placed in the reflection optical path of the second long-pass filter (22) for interference imaging; The second camera (16) and the second imaging lens (15) are placed in the reflection optical path of the full-wavelength semi-reflective and semi-transmissive filter (19) for fluorescence imaging; The working wavelength of the first laser source (7) is λ F , and the laser generated by it is reflected by the dichroic long-pass filter (17) and enters the main optical path; the working wavelength of the second laser source (10) is λ R , and it is reflected by the all-wavelength semi-reflective and semi-transmissive filter (19) and enters the main optical path; The wavelength relationship satisfies the following formula: λ F <λ1 < λ2 < λ E <530nm < λ4 < λ3 < λ5 < λ R ; The measurement method includes: Step S11: Prepare K micro-groove devices (1) with different groove depths, fill the micro-grooves with a fluorescent staining medium (2), and attach the surface of the gold coating (3) of the glass slide (4) to the top surface of the grooves to form a standard fixed-thickness fluorescent staining film; c ​ Step S12: Turn on the second laser source (10) and the second camera (16), focus on and photograph the standard fixed-thickness fluorescently stained film to obtain K c fluorescent images; Step S13: Prepare K microgroove devices (1) with different microgroove depths. Contact any edge of the surface of the gold coating (3) on the glass sheet (4) with the bottom surface of the groove, and use the fluorescent staining medium (2) to fill the wedge-shaped gap formed between the glass sheet (4) and the microgroove to form a standard wedge-shaped fluorescent staining film. w ​ Step S14: Turn on the first laser source (7), the second laser source (10), the first camera (14), and the second camera (16), focus on the standard wedge-shaped fluorescence staining thin film, and take fluorescence images and interference fringe images; Step S15: Repeat Step S14 for all standard wedge-shaped fluorescently stained films to obtain K w fluorescent images and interference fringe images of the films; Step S16: Calculate the film thickness of any pixel (i, j) of the k-th interference fringe image using the optical interference method wherein, is the depth of the micro-groove at (i, jc); is the gray value at the edge of the micro-groove; is the number of extreme points at the edge of the micro-groove; r f is the refractive index of the fluorescent staining medium (2); jc is the pixel column coordinate of the position with the maximum thickness of the standard wedge-shaped fluorescent staining film of the micro-groove device (1), p i,j (k) is the number of gray extreme points between the pixel (i, j) and the pixel (i, jc) in the i-th row of pixels; wherein, p i,jc (k) represents the number of extreme points at the groove edge, which is 0, g i,j (k) is the gray value of the pixel (i, j), g i,j (k)max is the nearest gray maximum value to the pixel (i, j) in the i-th row of pixels, g i,j (k)min is the nearest gray minimum value to the pixel (i, j) in the i-th row of pixels; Step S17: For K w Repeat S16 for the interference fringe images to obtain the film thickness corresponding to all the interference fringe images; Step S18: For K = K c + K w fluorescent images, construct a system of linear equations for calibration coefficients: Among them, a i,j (n) is the coefficient of the nth order fitting term corresponding to pixel (i, j), N is the total order of the fitting formula, h i,j (k) is the film thickness corresponding to pixel (i, j) in the kth image, where K = K c +K w In the fluorescence image, the front K c The photo is of a standard fixed-thickness fluorescent dye film, the thickness is known, and the K w Zhang photographed a standard wedge-shaped fluorescent dye film, and the thickness was calculated according to the formula in S16; Step S19: Solve the calibration coefficient linear equations constructed in step S18 to obtain a fitting term coefficient table; Step S20: Use the same optical measurement system to take fluorescence photos of the thin film to be measured; Step S21: Use the fitting term coefficients calculated in step S19, and according to the relationship formula between the thin film thickness and the fluorescence intensity value, calculate the thin film thickness corresponding to each pixel (i, j): where h i,j is the thickness of the film to be measured at pixel (i, j), and g i,j is the gray value corresponding to the pixel (i, j) of the fluorescence image of the film to be measured.

2. The fluorescence staining film thickness measurement method of interference-fluorescence in-situ coupling according to claim 1, wherein λ1 = 490nm; λ2 = 500nm; λ3 = 625nm; λ4 = 600 nm; λ5 = 630 nm; λ F = 470 nm; λ R = 655 nm; λ E = 515 nm.

3. The fluorescence staining film thickness measurement method of interference-fluorescence in-situ coupling according to claim 1, characterized in that The first camera (14) and the second camera (16) are interconnected through a synchronous signal line (23) to achieve synchronous triggering.

4. The fluorescence staining film thickness measurement method of interference-fluorescence in-situ coupling according to claim 1, characterized in that The fluorescence module further includes a first mirror mount (6); the dichroic long-pass filter (17) and the first long-pass filter (18) are mounted on the first mirror mount (6).

5. The fluorescence staining film thickness measurement method of interference-fluorescence in-situ coupling according to claim 1, characterized in that The optical interference module further includes a second mirror mount (9); the full-wavelength semi-transmissive and semi-reflective filter (19) is mounted on the second mirror mount (9).

6. The fluorescence staining film thickness measurement method of interference-fluorescence in-situ coupling according to claim 1, wherein, The beam splitting module further includes a third mirror mount (12); the dichroic short-pass filter (20), the short-pass filter (21) and the second long-pass filter (22) are mounted on the third mirror mount (12).

Images