CN1862308A - modular scanning probe microscope - Google Patents

Abstract

A modular scanning probe microscope in combination with an inverted fluorescence microscope, comprising five modules: the first module is an inverted fluorescence microscope; the second module is a three-jaw approximation device; the third module is a scan head; the fourth module comprises a transparent sample stage and an X, Y, Z three-dimensional scanner; the fifth module is a laser micro-expansion module. The modular structure of the invention utilizes each part of modules to easily build scanning probe microscopes with different structures, thereby greatly reducing the cost for purchasing various instruments; the three-dimensional scanner is used for realizing the original function of a fluorescence inverted microscope, and can also realize the special scanning near-field optical microscope function of microscope objective lens irradiation and collection, the observation of a determined area and a determined target, the detection of a laser confocal microscope and other operation and processing work mainly based on a probe or a microscope by combining all modules.

Description

Modular Scanning Probe Microscope

technical field

The invention relates to a scanning probe microscope, in particular to a modular scanning probe microscope combined with a fluorescent inverted microscope.

Background technique

Scanning probe microscope (hereinafter referred to as SPM) is a surface measurement instrument with ultra-high spatial resolution, which is easy to use and can be used for nanometer detection, manipulation and processing. Most of the existing scanning probe microscopes adopt a fixed structure, so they are only suitable for some specific applications, such as some are only suitable for small sample scanning, and some are only suitable for opaque sample detection. The combination of SPM, fluorescent inverted microscope and three-dimensional scanning sample stage can accurately locate the target to be scanned on the sample and the position of the needle tip. The method of scanning the needle tip of the sample can ensure the high coincidence of the needle tip and the illumination spot when the illumination spot is small. Thereby improving the contrast of the test image. The luminescence of objects, the fluorescent labeling of objects, and the nature of objects to change light characteristics make optical microscopes one of the most important tools for life science and material science research, and inverted microscopes are the most powerful microscopes in optical microscopes. The combination with SPM will produce functionality that neither alone could produce.

In the scanning probe microscope (invention patent, patent number: USA005850038A) of the prior art combined with an optical microscope, as shown in Figures 1 and 2: the entire device consists of two parts: an SPM test element 114 and an inverted optical microscope 130. The SPM test element 114 includes a Z-direction scanning stage 116, a position detection system 118 (including a laser light source 120, a dichroic mirror 122, and a light spot position sensor 124), a cantilever support 126, a cantilever probe 128, and a sample stage 142. The microscope 130 includes an objective lens 132 , an eyepiece 134 , an image acquisition device 136 , a monitor 138 , a laser switch 150 , a spectroscopic device 166 and a mirror 168 . During operation, the Z-direction scanning stage 116 realizes the approach of the cantilever probe 128 to the sample stage 142 , and then starts sample scanning, and the light spot position sensor 124 detects the Z-direction displacement of the cantilever probe 128 during the scanning process. The observation of the measurement sample and the observation of the relative position between the cantilever probe 128 and the sample stage 142 are obtained through the monitor 138 , while the fluorescence observation is directly accomplished through the eyepiece 134 of the inverted optical microscope 130 . The technical structure and principle are simple and relatively easy to implement, but it obviously has the following deficiencies:

1. The simple combination of an inverted microscope and a scanning probe microscope can only complete the original basic functions of the two, and cannot perform special work according to different needs, and cannot reduce the cost of the instrument.

2. This technology does not have an independent rough approximation mechanism, and it is inconvenient to realize AFM and SNOM functions on the same rough approximation device by simply replacing the needle tip of the head, and then realize other functions based on SPM, such as laser confocal microscope detection, and Probe-based or microscope-based operations and processing, etc.

3. The three directions of XYZ of the sample table cannot be scanned, and the selection of the area to be scanned at the micron level cannot be realized. Due to the way of scanning the sample with the probe, it is not conducive to the accurate coincidence of the needle tip and the illumination spot, which reduces the contrast of the scanned image.

4. During fluorescence observation, the illumination light is not directly irradiated on the sample after being converged by the lens system, but the light transmitted through the sample by the unreflected part of the end of the micro-cantilever probe enters the objective lens, so that the entire field of view cannot be uniformly illuminated, and The viewing light is very weak, so fluorescence imaging is not ideal.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art, and provide a modular scanning probe microscope. The modular scanning near-field probe microscope combined with the fluorescence inverted microscope of the present invention is convenient to adjust and replace, and utilizes each part of the module and It is easy to build scanning probe microscopes with different structures, which greatly reduces the cost of purchasing various instruments; the sharing of three-dimensional scanners not only realizes the original functions of fluorescence inverted microscopes, but also realizes the special illumination and collection of microscope objective lenses by combining all modules. Scanning near-field optical microscopy (SNOM) capabilities, observation of defined areas and defined targets, laser confocal microscopy inspection, and some other probe-based or microscope-based operations and processing.

Technical solution of the present invention is as follows:

A modular scanning probe microscope, which consists of five modules: the first module is an inverted fluorescence microscope; the second module is a three-jaw approximation device, including stepping motors, 2 manual coarse adjustment structures, lighting LEDs and CCD ; The third module is a scanning head, the fourth module includes a transparent sample stage and X, Y, Z three-dimensional scanners; the fifth module is a laser microscope extension module, including a third reflector, a beam splitter, a third lens, A first pinhole, a fourth lens, a laser, a fifth lens, a second pinhole and a PMT detector;

The CCD camera of the first module and the objective lens system are combined together, and the output signal of the CCD camera is supplied to the monitor; the second module (2) is in contact with the fourth module through the shaft ends of the stepper motor and two manual coarse adjustment structures connection; the third module is different according to the scanning head used; the fourth module has a square through hole in the middle of the X, Y, Z three-dimensional flatbed scanner; the transparent sample stage is fixed on the X, Y, Z Above the square through hole of the Z-direction three-dimensional flatbed scanner, they are fixed together on the first module by screws; the distance from the objective lens system of the first module (1) to the probe tip is equal to the working distance of the first module, and the The illumination device, the objective lens system, the sample and the probe tip are coaxial, the fifth module is fixed under the objective lens system of the first module by screws, the laser beam output by the laser passes through the fourth lens, the first pinhole and the third lens, and becomes The relatively uniform collimated beam converges to a certain point of the sample on the transparent sample stage after passing through the beam splitter, the third mirror and the objective lens system, and the reflected light (or transmitted light, or fluorescence of stimulated radiation) at this point passes through the objective lens After the system is reflected to the detection optical path by the reflector and the beam splitter, the fifth lens focuses it on the second pinhole, is received by the PMT detector, and is input into the computer for storage.

The third module is that the scanning head is a scanning near-field optical microscope scanning head, or an atomic force microscope scanning head, or a curved non-aperture probe scanning head.

The scanning head of the scanning near-field optical microscope includes a probe tip.

The atomic force microscope (AFM) scanning head includes a micro-cantilever probe tip, a first lens, a first mirror, a second mirror and a second lens.

The curved non-aperture probe scanning head includes a curved non-aperture probe and a vibrating piezoelectric table.

The combination of the first module, the fourth module, and the fifth module can realize the detection function of the confocal scanning laser microscope; the combination of the first module, the second module, the third module scanning near-field optical microscope scanning head, and the fourth module can realize scanning The function of the near-field optical microscope; the combination of the first module, the second module, the atomic force microscope (AFM) scanning head of the third module and the fourth module can realize the atomic force microscope of the sample scanning tip; the first module, the second module The combination of the curved non-aperture probe scanning head of the third module and the fourth module can realize the special scanning near-field optical microscope of microscope illumination and collection mode.

Compared with prior art, technical effect of the present invention is as follows:

1. The present invention integrates each unit device of the scanning probe microscope into smaller modules with certain functions, and can conveniently use these modules to quickly build microscopes with different structures without destroying the original structure of the inverted microscope, so as to adapt to The needs of various functions greatly reduce the cost of purchasing various instruments;

2. The detachable micro-displacement scanning sample stage is used, and the XYZ three-dimensional scanner is used to replace the inverted microscope sample stage on the basis of not destroying the original structure, which can carry a heavy load, and can also guarantee a high load The resonant frequency will not affect the performance of the entire microscope system. Under the disturbance of the probe, through the XYZ three-dimensional scanner sample stage with a through hole in the middle, the sample scanning needle tip mode can be used to realize the special scanning near-field irradiation and collection of the microscope objective lens Optical microscope (SNOM) function;

3. As a module, the three-jaw rough approximation structure can carry the AFM head and the SNOM head, and on this basis, realize all the functions of the SPM, observe the morphology of the sample with high resolution and study other surface features;

4. The combination of optical microscope and XYZ three-dimensional scanner can realize the observation of the determined area and the determined target and the selection of the area to be scanned at the micron level;

5. On the basis of the fluorescent inverted microscope, through the spectroscopic beam splitter and other self-designed optical components, adding laser beams and photomultiplier tubes with small holes to make it a laser confocal scanning microscope with a more miniaturized structure and fluorescence imaging more ideal.

Description of drawings

Fig. 1 is a schematic diagram of the principle of a scanning probe microscope combined with an optical microscope in the prior art.

Fig. 2 is a schematic diagram of the structure of a scanning probe microscope test element combined with an optical microscope in the prior art.

Fig. 3 is the schematic diagram of the principle of the modular scanning probe microscope combined with the fluorescence inverted microscope of the present invention

Fig. 4 is a schematic structural diagram of the principle structure of the fifth module laser microscope extension module of the present invention

Figure 5 is a schematic diagram of the principle of Embodiment 1 of the present invention

Figure 6 is a schematic diagram of the principle of Embodiment 2 of the present invention

Figure 7 is a schematic diagram of the principle of Embodiment 3 of the present invention

Figure 8 is a schematic diagram of the principle of Embodiment 4 of the present invention

Detailed ways

Please refer to Figure 3 and Figure 4 first, Figure 3 is a schematic diagram of the principle of the modular scanning probe microscope combined with a fluorescence inverted microscope of the present invention, and Figure 4 is a schematic structural diagram of the fifth module of the laser microscope extension module of the present invention. As can be seen from the figure, the modular scanning probe microscope combined with the inverted microscope of the present invention is made up of five modules: the first module 1 includes an illumination device 101, eyepiece tube 102, objective lens system 103, digital camera 104 and CCD camera 105; The second module 2 includes a stepper motor 201, two manual coarse adjustment structures 202, lighting LED203 and CCD204; the third module 3 is a scanning head, one of which is a scanning near-field optical microscope (SNOM) scanning head, including a probe tip 301 , the second is an atomic force microscope (AFM) scanning head, including a microcantilever probe tip 302, a first lens 303, a first mirror 304, a second mirror 305 and a second lens 306, and the third is a curved non-aperture probe The needle scanning head includes a curved non-aperture probe 307 and a vibrating pressure stage 308; the fourth module 4 includes a transparent sample stage 401 and X, Y, and Z three-dimensional scanners 402; the fifth module 5 is a laser microscope extension module, including The third mirror 501, the beam splitter 502, the third lens 503, the first pinhole 504, the fourth lens 505, the laser 506, the fifth lens 507, the second pinhole 508 and the PMT detector 509;

The CCD camera head 105 of the first module 1 and the objective lens system 103 are combined together, and the output signal of the CCD camera head 105 is supplied to a monitor (not shown); The shaft end of the structure 202 is in contact with the fourth module 4; the third module 3 is different according to the scanning head used; there is a square channel in the middle of the X, Y, Z direction three-dimensional flatbed scanner 402 of the fourth module 4. holes; the transparent sample stage 401 is fixed above the square through hole of the X, Y, and Z three-dimensional flatbed scanner 402 by bonding, and then is fixed on the first module 1 by screws together; the objective lens system 103 of the first module 1 reaches the probe The distance of the needle tip is equal to the working distance of the first module 1, the illumination device 101, the objective lens system 103, the sample and the probe tip of the first module 1 are coaxial, and the fifth module 5 is fixed under the first module 1 objective lens system 103 by screws , the laser beam output by the laser 506 passes through the fourth lens 505, the first pinhole 504 and the third lens 503 to become a relatively uniform collimated beam, and passes through the beam splitter 502, the third mirror 501 and the objective lens system 103 Converging at a certain point of the sample on the transparent sample stage 401, the reflected light, or transmitted light, or the fluorescence of stimulated radiation at this point is reflected to the detection optical path by the third reflector 501 and the beam splitter 502 after passing through the objective lens system 103, and then The fifth lens 507 focuses it on the second pinhole 508, is received by the PMT detector 509, and is input to a computer (not shown) for storage.

The third module (3) is that the scanning head is a scanning near-field optical microscope scanning head, or an atomic force microscope scanning head, or a curved non-aperture probe scanning head.

The scanning head of the scanning near-field optical microscope includes a probe tip 301 .

The atomic force microscope (AFM) scanning head includes a micro-cantilever probe tip 302 , a first lens 303 , a first mirror 304 , a second mirror 305 and a second lens 306 .

The curved non-aperture probe scanning head includes a curved non-aperture probe 307 and a vibrating piezoelectric stage 308 .

Example 1

The schematic diagram of the principle of Embodiment 1 is shown in FIG. 5 , which adopts the combination of the first module 1 , the fourth module 4 and the fifth module 5 . The first module 1 uses Nikon’s TE2000 universal biological microscope for research. This microscope has multi-port output design, ultra-precise Z-axis direction control and NIKON’s unique CFI60 optical system, and is equipped with new motorized accessories. Optional light sources and other accessories can be added to the far light path. The stray light elimination mechanism effectively blocks stray light and eliminates the influence of the surrounding temperature on the microscope prism, and has high anti-vibration performance. The fourth module 4 is a scanner 402 in xyz direction using P-733.3DD device of PI Company, the scanning range is 30×30×10 μm, the center of the device is a 50×50 cm light hole, and the no-load resonance frequency is 1200 Hz. The sample is placed on the transparent sample stage of the fourth module 4, and the sample is driven by the fourth module 4 to scan, and the laser beam output by the laser 506 passes through the fourth lens 505, the first pinhole 504 and the third lens 503, and becomes a relatively The uniform collimated light beam is converged at a certain point of the sample on the transparent sample stage 401 after passing through the beam splitter 502, the third reflecting mirror 501 and the objective lens system 103, and the reflected light at this point is passed through the objective lens system 103 and then is transmitted by the third reflecting mirror 501 and the objective lens system 103. The beam splitter 502 is reflected to the detection optical path, focused by the fifth lens 507 on the second pinhole 508, received by the PMT detector 509, and input to the computer for storage. Because the scanner of the fourth module 4 has a light hole, a two-dimensional tomographic image of a certain layer of the object is obtained by two-dimensional scanning, and then a large number of tomographic images are obtained by axial scanning, and a three-dimensional stereoscopic image is synthesized by computer image reconstruction. The scanning device is also controlled by a computer, which can realize the detection function of the confocal scanning laser microscope.

Example 2

Embodiment 2 is a scanning near-field optical microscope. The schematic diagram of the principle is shown in FIG. 6 , and the scanning head of the scanning near-field optical microscope is combined with the first module, the second module, and the third module, and the fourth module. The stepping motor 201 of the second module 2 adopts M-235.5DG of PI Company, and its one-way repeatability accuracy is 100nm; the lighting LED is fixed on the second module 2, and is adjusted to irradiate the probe tip at a certain angle. The third module 3 is fixed on the second module 2, and the probe tip 301 is perpendicular to the surface of the sample. When working, the first module 1 completes the real-time observation of the needle tip and the sample at the micron level, the second module 2 completes the rough approximation, and the fourth module 4 completes the fine approximation and performs feedback tasks, and at the same time completes the scanning function.

Example 3

The schematic diagram of the principle of Embodiment 3 is shown in Figure 7. The first module, the second module, the third module scanning near-field optical microscope scanning head, and the fourth module are combined. The scanning near-field optical microscope scanning head includes a microcantilever The probe tip 302, the first lens 303, the first mirror 304, the second mirror 305 and the second lens 306 place the sample on the transparent sample stage of the fourth module 4, and the third module 3 is driven by the second module 2 The sample is roughly approximated, and the X, Y, and Z three-dimensional flatbed scanners of the fourth module 4 perform fine approximation and scanning of the sample, thus forming an atomic force microscope with a sample scanning tip, whose illumination light can be irradiated from the sample. , it can also be a transmission type in which the illumination light is irradiated from below the sample, because the scanner of module four has a light-through hole.

Example 4

The principle schematic diagram of Embodiment 4 is shown in Figure 8, using the combination of the curved non-aperture probe scanning head and the fourth module using the first module 1, the second module 2, and the third module 3, and the 101 in the first module 1 Micromanipulation equipment can be installed to carry out microscope illumination to the fourth module 4, because the scanner of module 4 has a light hole, so can use 103 in the first module to carry out fluorescence signal collection, remove the illuminating LED203 and 203 in the second module 2 CCD204, the third module 3 uses a curved non-porous probe 307 and a vibrating pressure station 308. The sample is placed on the transparent sample stage of the fourth module 4, and the third module 3 is driven by the second module 2 to make a rough approximation of the sample. , the X, Y, Z of the fourth module 4 approach the sample finely to the three-dimensional flatbed scanner, and the vibrating piezoelectric stage 308 in the third module 3 drives the non-porous probe 307 to vibrate only in the Tapping mode in the vertical direction during scanning, The sample is scanned by the three-dimensional flat-bed scanner of the fourth module 4, and the signal feedback is completed by the third module 3 and the fourth module 4 to control the distance between the sample probes, thus forming a special scanning near-field of microscope illumination and collection mode Optical Microscopy (SNOM).

Claims (5)

1. A modular scanning probe microscope is characterized in that it is made up of five modules: the first module (1) is an inverted fluorescence microscope; the second module (2) is a three-jaw approximation device, including a stepping motor (201 ), 2 manual coarse adjustment structures (202), lighting LEDs (203) and CCD (204); the third module (3) is a scanning head, and the fourth module (4) includes a transparent sample stage (401) and X , Y, Z three-dimensional scanner (402); the fifth module (5) is a laser microscope expansion module, including a third reflector (501), a beam splitter (502), a third lens (503), a first needle Aperture (504), fourth lens (505), laser (506), fifth lens (507), second pinhole (508) and PMT detector (509); The CCD camera (105) of the first module (1) and the objective lens system (103) are combined together, and the output signal of the CCD camera (105) is supplied to the monitor; the second module (2) passes through a stepping motor (201) And the shaft ends of the two manual coarse adjustment structures (202) are in contact with the fourth module (4); the third module (3) is different according to the scanning head used; the X of the fourth module (4) There is a square through hole in the middle of the three-dimensional flatbed scanner (402) in the directions of X, Y, and Z; It is fixed on the first module (1) by screws; the distance from the objective lens system (103) of the first module (1) to the probe tip is equal to the working distance of the first module (1), and the lighting device of the first module (1) (101), the objective lens system (103), the sample and the probe tip are coaxial, the fifth module (5) is fixed below the first module (1) objective lens system (103) by screws, and the laser beam output by the laser (506) After passing through the fourth lens (505), the first pinhole (504) and the third lens (503), it becomes a relatively uniform collimated light beam, which passes through the beam splitter (502), the third mirror (501) and the objective lens system (103) converges on a certain point of the sample on the transparent sample stage (401), and the reflected light (or transmitted light, or fluorescence of stimulated radiation) at this point is passed through the objective lens system (103) and then is reflected by the reflective mirror (501) and the analyzer. The beam mirror (502) is reflected to the detection optical path, focused on the second pinhole (508) by the fifth lens (507), received by the PMT detector (509), and input to the computer for storage. 2. The modular scanning probe microscope according to claim 1, characterized in that the scanning head of the third module (3) is a scanning near-field optical microscope scanning head, or an atomic force microscope scanning head, or a curved non- Aperture probe scan head. 3. The modular scanning probe microscope according to claim 2, characterized in that the scanning head of the scanning near-field optical microscope comprises a probe tip (301). 4. The modular scanning probe microscope according to claim 2, characterized in that the scanning head of the atomic force microscope (AFM) comprises a microcantilever probe tip (302), a first lens (303), a mirror (304), second mirror (305) and second lens (306). 5. The modular scanning probe microscope according to claim 2, characterized in that said curved non-aperture probe scanning head comprises a curved non-aperture probe (307) and a vibrating piezoelectric stage (308).

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