TWI467227B - Three-dimensional microscope device and method using the same - Google Patents

Description

Penetrating three-dimensional microscopic device and method

The invention relates to the field of microscopy technology related to detecting biotechnology materials and organisms, in particular, the relationship between light intensity variation and reflectance change, combined with critical angle and CCD capture image technology to measure the surface of a light transmissive object. Or internal topography.

At present, the measurement of the shape of the observed objects is mostly biased toward the measurement of the microscopic system, such as: biomedical measurement, semiconductor industry, etc., and strives to pursue the microscopic world of the nanometer level, and sees the microscopic organisms that are indistinguishable to the naked eye through the microscope. Since Hans Janssen and his son Zacharias Janssen in 1950, they have tried to place several lenses on a bobbin and found that the objects seen through the lenses were magnified. They invented the world's most primitive telescopes and microscopes, and opened the history of the microscope. The types of optical microscopy technology can be divided into two-dimensional structure measurement and three-dimensional structure measurement. Two-dimensional structure measurement can be divided into three types, Total Internal Reflection Fluorescence Microscope (TIRFM) (Reference [1] [2] [3]), which is an evanescent wave generated by total reflection. The fluorophore in a thin layer of hundreds of nanometers thick on the surface of the sample is excited. This technique has been applied by biologists to single-molecule fluorescence imaging. TIRFM can be divided into two types. One is to use the light to transmit total reflection (in the other direction of the objective lens, the excitation laser is introduced, and the laser enters the aqueous solution and the wave plate between the observation object to generate total reflection) The other is to use a high-NA objective lens for total reflection (the objective-type system laser source is introduced by the edge of the objective lens to produce total reflection and to produce internal total reflection fluorescence excitation). Yan Yiguang (Ref. [4]) applied total reflection fluorescence microscopy technology to the immediate detection and manipulation of protein molecules by integrating total reflection fluorescence microscopy and microelectromechanical process technology to carry out protein in physical environment. When changing, the total reflection light field is used to excite the fluorescence with an evanescent wave, so that the total reflection fluorescence microscopy has an observation function within a size range of 150 to 700 nm from the glass substrate, and the signal background ratio of the image is much higher than Other microscopic techniques; and the lateral size function was also verified by measuring 1 μm of fluorescent particles.

The Near Field Scanning Mircoscope (NSOM: Near Field Scanning Mircoscope) (Ref. [5][6][7][8]) is based on Synge in the United Kingdom in 1928 and O' Keefe in the United States in 1956. Optical measurements are made in the near field (within a distance less than the wavelength) to avoid interference with light fluctuations in the far field (greater than one wavelength distance). Zhuang Jiayu (Ref. [9]) also uses non-destructive detection of different samples using a scanning near-field optical microscope. The ultraviolet light or green laser is coupled into the fiber, and the scanning near-field optical microscope is used to observe the defects of the grown gallium nitride film on the patterned sapphire substrate. Successful use of scanning near-field optical microscopy and self-made fiber optic probes with different light sources and photodetectors has developed non-destructive near-field measurements that can be used to detect the growth of different energy gap materials in different types of wafers.

Confocal microscopy was proposed by M. Minsky (Ref. [10]) in 1957, but at the time there was no suitable source of light, so it only stayed in a purely theoretical state and was not taken seriously. It was not until the 1960s that lasers and computers came out that they gradually gained attention. In 1969, P. Davidovits and MD Egger (Ref. [11]) developed a scanning confocal microscopy system using lasers (Ref. [12]). Through decades of efforts, technology and optics have continued to improve. Confocal microdisplay technology has matured. The first timely imaging scanning confocal microscopy system was constructed by G.Q.Xiao and G.S.Kino (Ref. [13]).

Many scholars have been working on the improvement of confocal microscopy technology. Wang Zhiping (Ref. [14]) et al. [developed differential confocal microscopy, which greatly improved the longitudinal resolution, making optical resolution comparable to scanning probe microscopy. Than, and have a larger dynamic range. In 1990, Denk (Ref. [15]) et al. proposed two-photon microscopy. This technique is similar to the basic architecture of confocal microscopy, except that a pulsed laser that emits very high intensity photons in a short period of time is used to excite one fluorescent molecule with two longer wavelength photons at a time. The high contrast in the axial direction is the biggest advantage of two-photon microscopy.

Optical Coherence Tomography (OCT) (Ref. [16]) uses low-coherence light or ultra-short laser pulses. The optical tomography technology developed by the company mainly scans living tissue in a non-invasive way to obtain high-resolution internal structural tomographic images. It was first published by JG and Fujimoto in 1991.

Lai Jinfa (Ref. [17]) developed an angle-biased microscope with a numerical aperture of 0.65. Using a common path heterodyne interferometry combined with a surface plasma resonance angle measurement architecture, the NA value of 0.65 can be used to increase the measurement resolution. degree. The structure has the advantages of high stability, low environmental and temperature resistance, easy assembly and monitoring, and can be monitored by computer at any time. The sensitivity can reach S=0.100deg/nm, the longitudinal resolution is R=3nm, and the lateral resolution is about 0.3. Μm (at NA=0.65). Most of them use the phase method or generate interference fringes for measurement, which is more accurate, but also relatively higher in terms of cost. Therefore, the development of a lower cost, better precision measuring device is to change direction. Therefore, after cost considerations, the method of the present invention uses a strength method to achieve lateral displacement measurements.

Zhan Yuansheng (Ref. [18]) proposed a large-area reflection measurement method combined with CCD light intensity image analysis technology to measure the surface profile of the object to be tested, using angle bias and optical imaging technology, by means of optical inductance measurement array The technique of taking out an image, measuring its area size by its two-dimensional image, and converting it into a height value by changing its light intensity (reflectance variable) into a three-dimensional image. The Republic of China Patent No. 100107449, which is currently in the application, "Optical Strength Type Three-Dimensional Surface Topography and Microscopic Measurement Apparatus and Method".

A first object of the present invention is to provide a transmissive three-dimensional microscope device and method, which mainly utilizes a method of measuring light intensity combined with a critical angle and a CCD capture image technique, and does not need to pass through the object to be tested for opacity. Scanning and interference can instantly use the light intensity change of the object to be tested, and then convert the reflectance into a surface height change through the soft body function, and obtain a relatively complete three-dimensional microscopic image at a time, and have high measurement The advantages of sensitivity, measurement range and low cost.

A second object of the present invention is to provide a transmissive three-dimensional microscope apparatus and method capable of compensating and correcting nonlinear errors.

A third object of the present invention is to provide a transmissive three-dimensional microscope apparatus and method that overcomes the problem of generating dispersion using a low beam source.

In order to achieve the above object, the technical means adopted by the present invention comprises: a laser light source; a beam expander comprising a spatial filter and a lens for filtering out stray light of the laser light source exiting beam for the light beam to exit the lens And causing the divergent light to converge into parallel light; the objective lens group is configured to allow the penetrating beam to be focused by the objective lens group to the penetrable object to be tested, and the objective lens group outputs the beam that penetrates the object to be tested into a parallel beam, so that the object The measuring object can be imaged by the objective lens group; the polarizing plate is placed on the adjacent side of the objective lens group to adjust the polarization direction of the light beam; and the splitting light is used to split the laser light source into two beams of reflected light and transmitted light. An angle-correctable angle sensor for small angle sensing near a critical angle for reflecting the reflected beam at least twice; a first image capturing unit for using the angle sensor The reflected light beam captures the image of the object to be tested; the corner is provided to provide the total reflection effect; and the second image capturing unit is configured to penetrate the beam from the right angle To capture the image of the object to be tested.

The technical means of the invention is that the laser light source is directed to the permeable object to be tested as the test light, and the test light passes through the objective lens group of the microscope group, and the test object obtains the test two boundaries under the microscope. The amount of change in the intensity of the light, and the amount of change in the reflectance of the test light is obtained according to the amount of change in the intensity of the light, and the surface height of the object to be tested is calculated according to the amount of change in the reflectance, and the three-dimensional object under test is obtained. Morphology.

one. Technical concept of the invention

The invention mainly utilizes an objective lens group to improve the resolution, and uses a critical angle intensity detection technique combined with an image capturing technique to measure the fine surface contour of the transparent object to be tested. When the laser light source passes through the transparent object to be tested, the transmitted beam is slightly angularly offset due to the change in the height of the surface of the object to be tested, so that the angle sensor that is incident on the parallelogram deviates from the original critical angle. The angle of the light, resulting in a sensitive increase in the intensity of the emitted light. Therefore, the ratio of the surface height of the object to be tested to the intensity of the transmitted beam is proportional to the reflectance, so that the change in reflectance can be used to describe the surface topography of the object to be tested. Finally, two groups of first and second image capturing units can be used to extract the two sets of reflectivity of the image to analyze the reflectance change, and the three-dimensional surface microstructure of the object to be tested can be specifically described.

two. Specific embodiment of the present invention 2.1 Method for measuring the height of a sample to be measured by a three-dimensional microscope

The method for measuring the three-dimensional shape of the object to be tested by the microscope, wherein the laser light source is directed to the light-transmitting object to be tested as the test light, and the test light passes through the objective lens group of the microscope group, so that the The measured object obtains the change amount of the light intensity of the test two-border light under the microscope, and obtains the change amount of the reflectance of the test light according to the change amount of the light intensity, so as to calculate the surface height of the test object according to the change amount of the reflectance. , obtained the three-dimensional shape measured by the microscope.

2.1.1 Relationship between incident external angle and reflectivity of the angle sensor of the present invention

To illustrate the reflectivity relationship of the angle sensor, the angle sensor is a parallelogram 稜鏡 as a preferred embodiment, and P-polarized light is taken as an example to change the reflectance inside the parallelogram ,, if other When the polarized light is incident, the measurement sensitivity can be adjusted according to individual needs. Since the P-polarized light is reflected twice in the parallelogram, the reflectivity is more sensitive than that of the primary reflection, and a curve close to linear can be obtained. Then, the average value of the slope is taken, and there is a relative relationship between the slope and the reflectivity of the parallelogram 稜鏡 and the external incident angle θ of the parallelogram 稜鏡. First, we discuss how to find the reflectivity and the relationship between the reflectivity and the external incident angle θ of the parallelogram 稜鏡. As shown in Figure 1, the reflected light is reflected twice inside the parallelogram. Then, from the boundary conditions and Snell's law, the relationship between the reflectance of the two reflections and the angle θ ₁ of the incident angle is obtained, as shown in equations (1) and (1-1) and (1-2). Show:

among them,

First, the outer angle θ of Fig. 1 is set to a range of 4 to 10 degrees, and the formula (1) = R _{p 2} represents two reflections, then the refractive index n ₁ = 1.51509 (when the wavelength λ = 632.8 nm), the air refractive index n ₂ = 1. 0003 , substituting (1), (1-1), (1-2), after using MATLAB program simulation analysis, the result shown in Figure 2 is made, but in order to prove the curve simulation, the reflectance curve of the parallelogram is actually measured by Fig. 3. Therefore, it can be seen from Fig. 3 that the angle of change is most sensitive at an angle of around 5.58 degrees. When θ ₁ = θ _c (θ _c is the critical angle), the θ obtained by substituting (1-1) 5.583 degrees. The present invention is based on the reflectance of P-polarized light as the main parallelogram, and the reflectance change is more sensitive than the S-polarized light. Therefore, the intensity measurement using P-polarized light is a preferred embodiment. (If other poles are used instead, the sensitivity of the reflectance to the change in the incident angle θ will decrease.)

2.1.2 Relationship between surface height and reflectivity of the object to be tested

As shown in FIG. 4, if the object to be tested (10) is inside the substrate, the object (10) outside the object to be tested (10) is in the environment of glass (13), the refractive index is n ₁ , and the object (10) is refracted. The rate is n ₂ . Therefore, the deflection angle β can be derived from the following formula:

n ₂ sin ₂ = n ₁ sin θ ₁ (2)

As can be seen from equation (4), the deflection angle β is proportional to the internal offset angle α. Therefore, when we know that the object to be tested (10) is in the environment of glass (13), the refractive index is n ₁ , and the refractive index n _{2 of} the analyte (10) can be obtained by the formula by measuring the angle β. The internal offset angle α of the object to be tested.

It can be seen from Fig. 4 that the light is incident on the object to be tested (10). If the surface height of the object to be tested (10) has a change in dh , the optical path is shifted from the original path direction to form an angular shift of +β or -β. the amount. In the object to be tested (10), the laser light is incident on the object to be tested (10) and then refracted. If the surface of the object to be tested (10) is flat on both sides of the surface, the first interface (17) is incident and is refracted. When the second interface (19) is parallel to each other, the transmitted light does not have an angular offset (β=0 ^o ); if the object to be tested (10) is as shown in FIG. 4, the first and second interfaces (17) When an angle of ±α is generated between (19), the amount of change in the deflection angle of +β or -β is formed, respectively. Returning to equation (4), when the slope of interface 2 is positive, we define the alpha value as positive, and the opposite, the alpha value is negative. θ _{t 2} is the exit angle, n ₂ is the refractive index of the object to be tested, dx is the moving distance per unit scan, and dh is the amount of change in the surface height of the object to be tested relative to dx . It is known that β and α maintain almost a linear relationship. also

n ₀ γ≒ n ₁ β

γ=( n ₂ - n ₁ )α

As can be seen from equation (5)

Therefore, the amount of surface height change can be written as equation (7)

Dh =α dx (7)

Substituting (6) into (7) gives:

Therefore, it can be known that the angle is biased toward γ=( n ₂ - n ₁ )α, so the amount of change in the incident angle of 稜鏡 is d θ = γ, and the relationship between the height dh and γ is dh = α dx . Therefore, the surface contour h of the object to be tested (10) can be regarded as the sum of the height difference variation dh of the x- direction pixels on each unit interval dx , which can be written as equations (9) and (10):

h = B ( R _{P 2} - D ) (10)

D is a constant, B = BD is the initial height, n ₁ and n ₂ are the refractive indices of the substrate and the object to be tested, respectively, and R _{p 2} is the measured P-pole secondary internal reflectance when dX = Mdx and n ₂ < n ₁ , then B >0. dX can be regarded as the size of the pixel.

2.3 Structure of the three-dimensional microscope device of the present invention

Based on the foregoing method, as shown in FIG. 5, the three-dimensional microscope apparatus of the present invention comprises: a laser light source (20) which is a He-Ne Laser source having a wavelength of 632.8 nm for generating a laser beam; a dimmer (21) for attenuating the intensity of the laser beam emitted by the laser source (20); a beam expander (22) comprising a spatial filter and a lens (25) The spatial filter comprises a first objective lens (23) <its NA = 0.45> and a pinhole (24). When the laser beam is focused on the pinhole (24) through the first objective lens (23), At this time, the emitted light will filter out the influence caused by the stray light, thereby improving the beam precision, and the lens (25) is used to converge the divergent light passing through the spatial filter into a parallel beam; an objective lens group (26), The second objective lens (261) and the third objective lens (262) form a dual objective lens shape for the biological object to be tested (10) to be placed in the middle of the second objective lens (261) and the third objective lens (262). The test object (10) may be a single-celled organism such as an eye worm, a paramecium, or the like, and the light beam penetrating the lens (25) is focused by the second objective lens (261). On the object (10), the object to be tested (10) can be placed on a biaxial moving platform (27), and the two-axis moving platform (27) uses two single axes to move the platform for fixing the object to be tested (10). Making a slight lateral displacement, the object to be tested (10) can be moved to the set position, and then the third objective lens (262) outputs the beam that penetrates the object to be tested (10) into a parallel beam and is a test beam (this) When the object to be tested is used as the reference surface, the third objective lens (262) is driven by the uniaxial movement to make a slight longitudinal displacement, so that the object to be tested (10) can be clearly imaged; a polarizing plate (29) Placed on the adjacent side of the third objective lens (262) of the objective lens group (26) for adjusting the transmission axis angle of the test beam transmission to change the polarization direction of the test beam from the objective lens group (26); a beam splitter (30) for splitting the test beam from the polarizing plate (29) into a reflected beam (45) and a penetrating beam (40); an angle sensor (32), which is a parallelogram, in The critical angle is used for micro-angle sensing for at least two reflections of the penetrating beam (40). Under the laser source (20), the refractive index is 1.51509. The device (32) is disposed on a rotating platform (321) for facilitating the rotation angle sensor (32) to adjust the angle of incidence of the penetrating beam (40) to the angle of the critical angle; a first image capture a unit (33), which is a photo-electrical inductance measurement, such as a charge-coupling component (abbreviation: CCD), is used to extract a light beam emitted from the angle sensor (32) to obtain a first object (10) having a first a first image of light intensity, which faces the angle sensor (32); a constant angle 稜鏡 (35), thereby providing a function of the reflected beam (45) to produce total reflection, comprising two first waists that are isosceles ( 351), a second waist portion (352), and a sloped bottom surface (353) of a longest bottom surface, the first waist portion (351) faces the beam splitter (30), and the reflected light beam (45) is entered by the first waist portion (351). Total reflection is generated on the longest inclined bottom surface (353), and then emitted by the opposite second waist portion (352). Since only one total reflection occurs, the rotation of the image is changed, causing image inversion; a second image capturing unit (37) for photo-sensing, such as a charge-clamping component (abbreviation: CCD) for extracting the lens from the right-angle prism (35) The emitted light beam obtains a second image of the object to be tested having a second light intensity, in particular a second waist portion (352) facing the right angle 稜鏡 (35).

2.4 Measurement Example of Three-Dimensional Microscope Apparatus of the Present Invention

Referring to FIG. 5, the laser is used as the laser source (20), and the laser source (20) generates a laser beam incident beam expander (22) to expand the incident laser beam into a parallel beam. The second objective lens (261) is incident on the object to be tested (10), and then emitted by the third objective lens (262) as a test beam. The test beam passes through the polarizing plate (29) to form P-polarized light, and is incident on the spectroscopic beam ( 30), the test beam splits the light into two beams of the penetrating beam (40) and the reflected beam (45) via the beam splitter (30), and the penetrating beam (40) advances through the angle sensor (32), the user The rotating table (32) can be rotated until the incident angle of the penetrating beam (40) reaches a critical angle, and the position of the first image capturing unit (33) is appropriately adjusted to be imaged on the first image capturing unit (33). , set its light intensity distribution to I _{CCD 2} . The reflected beam (45) is transmitted through the first waist portion (351) through the right angle 稜鏡 (35), and is reflected by the oblique bottom surface (353), and then imaged in the second image capturing unit (37) to set the light intensity distribution. The picture shows I _{CCD 1} .

Secondly, the first image capturing unit (33) (37) is directly connected to the computer and the first and second image capturing units (33) (37) to display the images captured by the first and second image capturing units (33) (37), and the two images are overlapped. After taking out the light intensity ratio The reflectivity can be obtained, and finally analyzed by Matlab software (mathematical software) to depict the three-dimensional surface topography of the analyte (10) under the microscope.

Participation. Three-dimensional microscope device measurement embodiment of the present invention 3.1 for 20 lines/mm penetrating diffraction grating

In the experimental example of the present invention, the object to be tested is a 20 lines/mm transmissive diffraction grating, and through the structure of FIG. 5, when the total reflection is caused by the right angle 稜鏡 (35), the image is imaged by the second image capturing unit (37). The intensity map of the 20 lines/mm grating taken ( I _{CCD 1} ), the result is shown in Figure 1 of Annex 1, and the light near the critical angle is generated by the angle sensor (32), and imaged on the first image. The intensity distribution map ( I _{CCD 2} ) of the 20 lines/mm grating captured by the capture unit (33) is as shown in Figure 2 of Annex 1, and the reflectance information is obtained by completely overlapping the Figure 1 and Figure 2 of the Annex 1. The surface height map obtained by the formula of (10) h = B ( R _{P 2} - D ) is shown in Fig. 6.

Therefore, the data is extracted from Fig. 6, and the maximum height difference of the 360th column, the 420th column, and the 480th column in Fig. 6 is calculated to be about 84 nm, 81 nm, and 86 nm, respectively, and these conditions are as shown in Fig. 7. In this embodiment, about 10 columns are calculated and the total average height is 84 nm, as shown in FIG. Therefore, in Table 1 of Annex 3, it can be seen that the difference between the average value of the total average height difference of the y-axis and the height measured by the commercial film measuring machine (83.45 nm) is 0.55 nm, which means that the measurement of this embodiment is known. The percentage error is close to 1%. The measured result of the present invention using the measurement results of the present invention is very small compared with the measurement result of the commercial film measuring machine Dektak-6M, and the invention can be proved to be implementable.

3.2 Measurement of Euglena

The eye worm (Euglena) is a species of the genus Euglena in the organism. Euglena is a single-celled organism with a body length of about tens of microns, a wide middle, and narrow ends. The rear section is sharper and the front end is round. The whole body is a large cell, and a well-permeated outer membrane is wrapped outside to allow small molecules such as water and oxygen to pass through. Its refractive index is about 1.3. The method of implementation is as described above, and FIG. 1 and FIG. 2 of FIG. 2 show gray-scale patterns of light intensity changes taken by the second image capturing unit (37) (33) in the vicinity of the total reflection angle and the critical angle, respectively. After the overlap, the light intensity of each point is I _{CCD 1} and I _{CCD 2 respectively} , and the reflectivity R _p2 can be obtained to obtain the height morphology of the eye worm of the object to be tested. When shooting, referring to FIG. 5, it is necessary to adjust the object to be tested (10) to the confocal plane of the objective lens group (26), and adjust the positions of the first and second image capturing units (33) (37) so that Image analysis. After being converted into the surface height, the three-dimensional image presented is as shown in FIG. 9, and is enlarged and observed as shown in FIG. The actual size of the ophthalmite measured according to the present invention is 70 μm × 35 μm × 0.87 μm.

3.2 Applicable to the measurement of Paramecium

The practice is shown in the measurement of Euglena, and all measurements of the paramecium are not repeated. The actual size of the paramecium obtained in this experiment is 390 μm × 150 μm × 0.7 μm, and its three-dimensional image is shown in Fig. 11.

Hey. in conclusion

Therefore, with the above technical features, the present invention does have the following features:

1. The invention utilizes the image capturing unit of the photo-sensing function to extract the image of the object to be tested, thereby converting the light intensity or reflectance of each point into a measuring device for the surface height of the object to be tested, thereby making the microscopic observation The object can be used to derive a three-dimensional image, so the present invention has the advantage of non-contact, non-destructive, non-interferometric methods.

2. The invention is a transmissive optical system measurement, the lateral resolution can reach at least 0.5 μm, and the longitudinal resolution is at least a few nanometers, so the measurement can be applied for a wide range of applications, such as biotechnology, biomedical testing, Film thickness measurement, optoelectronic industry, semiconductor industry, precision manufacturing, food processing industry, chemical industry, etc. can be applied to industries that require fine observation and measurement; even for defects, defects, surface analysis, film thickness, and biology Sample or roughness measurement.

3. The invention can obtain information of a whole surface to be tested without scanning, and the measurement range and the magnification can be adjusted.

4. The polarization direction of the laser light source used in the present invention can be changed according to the needs of the user, and is not fixed.

5. The present invention performs three-dimensional microscopic measurement of a microscope for a light-transmitting object to be tested.

6. The present invention comprises two sets of image capturing units for photo-sensing, one for the light to enter the total reflection angle and the other for the light entering the critical angle, so that the adjustment angle can be reduced without adjusting the total reflection angle. The error is effectively compensated and corrected.

7. According to the invention, the lateral resolution can be higher than 0.5 micron, the longitudinal resolution can be higher than several nanometers, and the resolution and sensitivity are better.

The above is only one of the possible embodiments of the present invention, and is not intended to limit the scope of the patents of the present invention, and the equivalent implementations of other changes according to the contents, features and spirits of the following claims are It should be included in the scope of the patent of the present invention. The invention is specifically defined in the structural features of the request item, is not found in the same kind of articles, and has practicality and progress, has met the requirements of the invention patent, and has filed an application according to law, and invites the bureau to approve the patent according to law to maintain the present invention. The legal rights of the applicant.

(10). . . Analyte

(13). . . glass

(15). . . air

(17). . . First interface

(19). . . Second interface

(20). . . Laser source

(twenty one). . . Dimmer

(twenty two). . . Beam expander

(twenty three). . . First objective

(twenty four). . . Pinhole

(25). . . lens

(26). . . Objective lens group

(261). . . Second objective

(262). . . Third objective

(27). . . Two-axis mobile station

(28). . . Single axis mobile station

(29). . . Polar plate

(30). . . Splitter

(32). . . Angle sensor

(321). . . Rotary table

(33). . . First image capturing unit

(35). . . Right angle 稜鏡

(351). . . First waist

(352). . . Second waist

(353). . . Oblique bottom

(37). . . Second image capturing unit

(40) (45). . . beam

Figure 1 is a schematic illustration of the two embodiments of the present invention in which the light is reflected twice in a parallelogram.

Fig. 2 is a graph showing the change of the reflectance of the P-polarized double reflection of the present invention to the external angle.

Fig. 3 is a graph showing the reflectance measurement of the parallelogram of the present invention.

Figure 4 is a schematic view showing the relationship between the surface height and the deflection angle of the present invention.

Fig. 5 is a schematic view showing the structure of an embodiment of the intensity-type transmissive three-dimensional microscope of the present invention.

Fig. 6 is a measurement 3D diagram of a 20 lines/mm grating of the first measurement example of the present invention.

FIG. 7 is a comparison diagram of the 360th, 420, and 480 list surface contours measured by the present invention according to FIG. 6.

Figure 8 is a graph showing the average height of the surface profile of the present invention as measured in Figure 6.

Fig. 9 is a third measurement example of the present invention, and an eye worm measurement 3D map.

Figure 10 is a partial view of the eye worm obtained by Figure 9 of the present invention.

Figure 11 is a third comparative measurement of the present invention, a 3D map of the paramecium measurement.

Attachment 1 is a plan view of the image taken by the CCD when the external angle is at the total reflection angle; and FIG. 2 is a 20 lines/mm grating of the present invention when the external angle is near the critical angle. The image plan taken by the image capture unit.

Attachment 2: Figure 1 is a measurement example 2 of the present invention. The object to be tested is an eye worm. The image of the CCD is taken when the external angle is at the total reflection angle. Figure 2 is a measurement example 2 of the present invention: the object to be tested is an eye worm A plan view of the image taken by the CCD when the angle of the outer corner is near the critical angle.

Attachment 3: Height measurement comparison table for 20 lines/mm grating.

Annex 4: References

(10). . . Analyte

(20). . . Laser source

(twenty one). . . Dimmer

(twenty two). . . Beam expander

(twenty three). . . First objective

(twenty four). . . Pinhole

(25). . . lens

(26). . . Objective lens group

(261). . . Second objective

(262). . . Third objective

(27). . . Two-axis mobile station

(28). . . Single axis mobile station

(29). . . Polar plate

(30). . . Splitter

(32). . . Angle sensor

(321). . . Rotary table

(33). . . First image capturing unit

(35). . . Right angle 稜鏡

(351). . . First waist

(352). . . Second waist

(353). . . Oblique bottom

(37). . . Second image capturing unit

(40) (45). . . beam

Claims (10)

A transmissive three-dimensional microscope device comprising: a laser light source for emitting a laser beam; a beam expander comprising a spatial filter and a lens for filtering out the laser beam Astigmatism, and exiting to the lens to converge into a parallel beam; an objective lens group for focusing the parallel beam on a penetrable object to be tested, and outputting the beam penetrating the object to be tested in parallel for testing a polarizing plate for adjusting a polarization direction of the test beam; a splitting beam for dividing the test beam into a reflected beam and a penetrating beam; an angle-adjustable angle sensor, Having a small angle sensing near the critical angle for the reflected beam to be reflected at least twice; a first image capturing unit for extracting the object to be tested from the beam emitted from the angle sensor a first image having a first light intensity; a constant angle 稜鏡 for providing a total reflection of the reflected light beam; and a second image capturing unit for emitting a light beam from the right angle 撷Taking the object to be tested has a second light intensity A second image. The transmissive three-dimensional microscope device of claim 1, wherein the angle sensor is a parallelogram for the reflected beam to be reflected twice, and the angle sensor is coupled to a rotatable angle. Rotating table. The transmissive three-dimensional microscope device of claim 1, wherein the laser source is a He-Ne laser source having a wavelength of 632.8 nm between the laser source and the beam expander A dimmer is disposed to attenuate the intensity of the laser light source; the spatial filter of the beam expander includes a first objective lens and a pinhole, and the laser light source is focused on the pinhole through the first objective lens. The transmissive three-dimensional microscope device of claim 1, wherein the objective lens group comprises a second objective lens and a third objective lens to form a dual objective lens shape, wherein the object to be tested is placed on the second objective lens and the third objective lens intermediate. The transmissive three-dimensional microscope device according to claim 4, wherein the object to be tested is placed on a biaxial moving platform for fixing the object to be tested for slight lateral displacement, and the polarizing plate is disposed The adjacent side of the third objective lens of the objective lens group. The transmissive three-dimensional microscope device of claim 1, wherein the right angle 稜鏡 includes two first waist portions and a second waist portion, and a slanted bottom surface; the first waist portion faces the astigmatism The penetrating beam enters from the first waist portion, and total reflection is generated on the oblique bottom surface, and the light beam is emitted from the second waist portion. A penetrating three-dimensional microscope measuring method, which provides a penetrating three-dimensional microscope device according to claim 1, wherein a laser beam is generated by the laser light source; and the laser beam is filtered by the beam expander And the exiting light converges to a parallel beam; the parallel beam is focused by the objective lens on a penetrable object to be tested, and the beam penetrating the object to be tested is outputted in parallel as a test beam; The polarizing plate adjusts a polarization direction of the test beam; the test beam is divided into a reflected beam and a penetrating beam by the beam splitter; and the angle sensor is used for small angle sensing near a critical angle, Providing the penetrating beam at least twice; and extracting, by the first image capturing unit, the first image of the object to be tested having a first light intensity from the beam emitted from the angle sensor;稜鏡 providing a total reflection of the reflected light beam; and extracting, by the second image capturing unit, the light beam emitted from the right angle 稜鏡 to capture a second image of the object to be tested having a second light intensity; Calculating the software according to the first light intensity The ratio of the reflectance of the test beam is determined by the ratio of the second light intensity, and the surface height of the object to be tested is calculated according to the amount of change in the reflectance, and the three-dimensional surface topography of the object to be tested is drawn. The method of claim 7, wherein the angle sensor is a parallelogram for the reflected beam to be reflected twice, and the angle sensor is coupled to a rotating table. The method of claim 7, wherein the objective lens group comprises a second objective lens and a third objective lens to form a dual objective lens shape, the test object being placed between the second objective lens and the third objective lens. The method of claim 7, wherein the object to be tested is placed on a biaxial moving platform for the micro lateral displacement of the object to be tested.