Nanoscale morphology contrast characterization method for SEM under ultralow voltage and short working distance

Abstract

The invention discloses a nanoscale morphology contrast characterization method under an SEM ultra-low voltage and a short working distance, and relates to the technical field of material surface microstructure detection and analysis, a sample is placed on a sample table, and a target area is selected in a TV mode; switching to an imaging mode, and performing astigmatism correction and focusing under the conditions of conventional acceleration voltage and working distance; switching back to the TV mode, and shortening the working distance to be lt; 5 mm; switching to the imaging mode again, reducing the acceleration voltage to be less than or equal to 1kV, and focusing and imaging the same area; and repeating the steps, and carrying out system imaging under different parameter combinations of ultralow voltage and short working distance so as to obtain nanoscale morphology contrast information of the sample metasurface. According to the invention, through the cooperation of ultra-low voltage and short working distance, the nondestructive and high-contrast visualization of the surface nanoscale native morphology can be realized without metal spraying, and the technical problems that the surface signal is averaged and the charge damage is serious under the deep nanoscale of the traditional SEM are solved.

Description

Technical Field

[0001] This invention relates to the field of material surface microstructure detection and analysis technology, and in particular to a method for characterizing nanoscale morphology contrast under ultra-low voltage and short working distance of SEM. Background Technology

[0002] With the rapid development of cutting-edge technologies, the structural features of systems such as metasurfaces, two-dimensional quantum materials, nanocatalysts and extracellular vesicles have entered the "deep nano" scale (≤20nm). The 1-3nm level undulations, defects or functional sites on their surfaces directly determine the optical resonance performance of materials, the activity efficiency of catalysts and the recognition specificity of biological particles, which are the core factors affecting technological breakthroughs in related fields.

[0003] In the field of materials, the performance of advanced devices such as superlenses and achromatic metasurfaces is highly dependent on step errors of less than 2 nm and lateral feature precision of less than 10 nm. Any tiny deviation in surface roughness can lead to a sharp drop in device focusing efficiency or even failure. In the field of chemistry, the active sites of single-atom catalysts are only exposed at defects on the surface of the support. If these sites cannot be accurately located with nanoscale contrast, the modeling of the structure-activity relationship of the catalyst will lose its experimental benchmark, which will seriously restrict the explanation of the catalytic mechanism and the optimization of catalyst performance. In the field of biomedicine, the difference in the protein canopy layer of exosome membranes of less than 5 nm is the key to determining its binding specificity with receptor cells. However, conventional detection methods often mask this core information, which directly affects the accurate identification of early disease diagnostic biomarkers.

[0004] Traditional scanning electron microscopy (SEM) typically employs conventional accelerating voltages of 5 kV or higher and working distances of ≥8 mm when examining the aforementioned systems. Under these conditions, electrons can be injected into the sample to depths of tens of nanometers. A large portion of the collected signals originates from the subsurface layer of the sample, resulting in the "averaging" or even complete obliteration of the true nanoscale morphology, making accurate characterization impossible. Furthermore, high-dose electron irradiation can easily induce problems such as sample charging, thermal damage, and carbon contamination. For sensitive samples such as polymer carriers, biological soft tissues, or low-dimensional materials, irreversible structural degradation often occurs during imaging, rendering them unusable for original-state detection.

[0005] To mitigate the charging problem, existing technologies often employ metal sputtering for sample pretreatment. However, this method introduces artificial artifacts into the sample surface, completely obscuring key surface features at the 1-3 nm level, which contradicts the requirements for "deep nanoscale" detection. More critically, the industry has yet to develop an ultra-low voltage, short working distance SEM imaging method that eliminates the need for metal sputtering, offers controllable parameters, and provides repeatable results. This makes it impossible to accurately characterize the nanoscale morphology contrast of metasurfaces, single-atom catalysts, and bio-nanoparticles in their original state, becoming a common technological bottleneck connecting the three major fields of precision material processing, catalytic mechanism elucidation, and bio-nanodiagnostics. Summary of the Invention

[0006] To address the problems of large electron injection depth, surface signal distortion, severe sample damage, and poor cross-domain applicability in the characterization of samples at the "deep nanoscale" scale, the present invention provides a nanoscale morphology contrast characterization method under ultra-low voltage and short working distance of SEM. By precisely controlling the accelerating voltage, working distance, and detector mode, it can achieve accurate visualization of the original morphology of the sample surface at the 1-3nm level without the need for metal sputtering, while avoiding sample damage caused by excessive electron dose, and has universality across the fields of materials, chemistry, and biomedicine.

[0007] The technical solution provided by this invention is: A method for characterizing nanoscale morphology contrast under ultra-low voltage and short working distance SEM, the method comprising: S1: Place the sample on the SEM sample stage and select the target imaging area in TV mode; S2: Switch the SEM from TV mode to imaging mode, select the In-lens secondary electron detector, and perform astigmatism correction and focusing under normal accelerating voltage and working distance conditions; the normal accelerating voltage is greater than or equal to 5kV, and the working distance is greater than or equal to 8mm. S3: Switch the SEM from imaging mode back to TV mode, and shorten the working distance to the target short working distance in the visible state; the target short working distance is less than 5mm; S4: Switch the SEM from TV mode back to imaging mode, select the In-lens secondary electron detector, reduce the accelerating voltage to an ultra-low accelerating voltage, and focus on the same target area; the ultra-low accelerating voltage is less than or equal to 1kV. Repeat steps S3 and S4 to perform system imaging under various parameter combinations of ultra-low accelerating voltage and short working distance, in order to obtain and compare nanoscale morphological contrast information of the sample metasurface.

[0008] Furthermore, the target imaging region comprises grains with two or more crystal orientations.

[0009] Furthermore, during the process of changing the accelerating voltage and / or working distance for system imaging, the electron beam current and electron beam irradiation area remain constant.

[0010] Furthermore, the various parameter combinations of the ultra-low acceleration voltage and short working distance are systematically screened using single-factor experimental methods, orthogonal experimental methods, or a combination of both.

[0011] Furthermore, the ultra-low accelerating voltage is 0.1kV to 1.0kV.

[0012] Furthermore, the short working distance is 0.5mm to 5.0mm.

[0013] Furthermore, the optimized combination of ultra-low acceleration voltage and short operating distance includes: A combination of 0.3kV-0.7kV and 0.5mm-1.0mm; or a combination of 0.5kV-1.0kV and 2.5mm-3.5mm.

[0014] Furthermore, the electron beam current is fixed at 51 pA, and the single-frame electron dose is 6.5 e. - / nm 2 The total dose shall not exceed 830e - / nm 2 .

[0015] Furthermore, the sample is a conductive or semi-conductive material that does not require metal sputtering, including graphene, metasurface materials, single-atom catalysts, or bio-nanoparticles.

[0016] Furthermore, the method also includes performing contrast quantitative analysis on the acquired SEM images to calculate the surface-subsurface mixing information index CV (Contrast Value), the formula of which is: CV=( I - I 0) / I 0×100%, in, I 0 represents the average grayscale value of the image in the substrate region. I This represents the average grayscale value of the image for the surface feature region.

[0017] The present invention has the following technical effects: 1. Achieved non-destructive, high-contrast visualization of the native nanoscale morphology of sample metasurfaces. By compressing the information collection depth to 1-3nm under ultra-low voltage (≤1kV) and short working distance (<5mm), and combining it with the high surface sensitivity of the In-lens detector, this invention can directly reveal the original features of the surface steps, defects and protein crowns at the 1-3nm level without metal sputtering, thus truly reflecting the original state of the sample.

[0018] 2. A repeatable and scalable systematic imaging method and parameter system were established. This invention systematically explores the synergistic relationship between "ultra-low voltage, short working distance, and in-lens detector," providing an experimentally validated optimal parameter matrix. Through a clearly defined mode-switching procedure and experimental method, it elevates previous sporadic, experience-based attempts into a systematic and standardized imaging approach, ensuring the reliability and universality of the results.

[0019] 3. It achieves precise control of electron dosage and possesses excellent non-destructive imaging capabilities. By fixing the beam current and controlling the integration time, this invention limits the total electron dose to a low level, below the damage threshold of sensitive materials, thereby enabling repeated imaging of the same area without significant charging, contamination, or structural damage.

[0020] 4. It possesses cross-domain versatility, solving common technical bottlenecks across multiple disciplines. This method has wide applicability. The same technical solution can be directly applied to fields such as materials (metasurfaces), chemistry (single-atom catalysts) and biomedicine (exosomes) without any fundamental modification, solving common problems in the characterization of nanoscale surface morphology. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the internal structure of the Sigma 360 scanning electron microscope selected in this embodiment of the invention; Figure 2 The Graphene / Cu sample of this embodiment of the invention was subjected to conventional V acc SEM images of the target area on the lower surface of the WD; Figure 3 These are a series of SEM images of the Graphene / Cu sample from an embodiment of the present invention, corresponding to different accelerating voltages and working distances. Figure 4 These are SEM images of the Graphene / Cu sample from an embodiment of the present invention under ultra-low accelerating voltage and short working distance and conventional accelerating voltage and working distance, wherein... Figure 4 a is the SEM image under the conditions of Vacc=0.3kV and WD=0.5mm. Figure 4 b is the SEM image under the conditions of Vacc=1.0kV and WD=3.5mm. Figure 4 c represents the SEM image under the conditions of Vacc=5.0kV and WD=7.1mm; Figure 5 SEM images of grain regions with different crystal orientations in the Graphene / Cu sample of this invention at ultra-low accelerating voltage and short working distance; Figure 6 This is a flowchart of the method of the present invention. Detailed Implementation

[0022] To make the objectives, advantages, and features of the present invention more apparent, the following description is provided in conjunction with the appendix. Figure 1-6 The present invention will be further described in detail below with reference to specific embodiments.

[0023] Example 1 like Figure 6 As shown, a metasurface nanoscale morphology contrast scanning electron microscopy imaging method based on ultra-low voltage and short working distance includes: Place the sample on the SEM sample stage (chamber) and select the target area in SEM TV mode; Based on the target area selected in SEM TV mode, switch SEM to imaging mode and select In-lens secondary electron (SE) detector mode; Firstly, at the conventional accelerating voltage (V) acc Astigmatism cancellation and imaging focusing are performed under imaging conditions of 5kV and above and working distance (WD=8mm); Based on the first selected imaging mode and parameters (V) acc ,WD), to take scanning electron microscope images of the target area; Switch the SEM back to TV mode. In the visible state, adjust the working distance to the target distance using Stage Z. During the sample stage rise, closely monitor the distance between the electron gun and the sample to prevent damage to the electron gun and contamination or damage to the sample surface caused by impact. Switch the SEM back to In-lens detector imaging mode, and under the selected working distance, select different accelerating voltages to focus on the target area and take scanning electron microscope images. Adjusting the accelerating voltage (V) acc While maintaining the working distance (WD), other parameters of the scanning electron microscope, such as electron beam current (I) and irradiated area (A), remain unchanged, and SEM imaging is performed in In-lens detector mode; SEM imaging was performed using single-factor and orthogonal experimental methods under the imaging parameter combinations shown in Table 1. Table 1 V used for SEM imaging acc Combined with WD parameters

[0024] After each SEM image is captured for the accelerating voltage, the SEM needs to be switched to TV mode for working distance adjustment. When imaging the target area, it needs to be switched back to In-lens detector mode.

[0025] The sample used for imaging was a copper foil with a partial surface area covered by a single layer of graphene islands, i.e., incompletely covered copper-based graphene (Graphene / Cu).

[0026] One of the selected target regions should contain grains with two or more crystal orientations.

[0027] The standard accelerating voltage selected for the first imaging was 5.0 kV.

[0028] The initial imaging was performed at a working distance of 8.0 ± 1.0 mm.

[0029] After adjusting the working distance, the selected target working distances are 0.5, 1.0, 1.5, 2.0, 2.5, 3.5, and 4.5-5.5 mm, respectively.

[0030] Among them, the selected accelerating voltage (V acc The voltage ratings are 0.1, 0.3, 0.5, 0.7, and 1.0 kV, respectively.

[0031] By precisely adjusting the accelerating voltage and working distance, and employing two parameter ranges that are generally not involved in traditional imaging methods, a model relating accelerating voltage, working distance, electron beam penetration depth, and the amount of secondary electrons collected is used to obtain nanoscale morphological contrast of sample surfaces at ultra-low accelerating voltage and working distance. This provides a powerful technical method for overcoming common bottlenecks in nanoscale characterization and precise processing of material surfaces / interfaces, elucidation of catalytic mechanisms, and bio-nanodiagnostics.

[0032] Example 2: Take ultra-low voltage-short working distance SEM imaging of nanoscale steps on copper-based graphene (Graphene / Cu) metasurface as an example.

[0033] 1. Sample Preparation Commercial graphene / Cu samples were used, with the following parameters for the single-crystal copper foil: Alfa, 99.8%, thickness 25µm. Single-layer discontinuous graphene islands were obtained: CVD growth at 1050°C, 10 sccm CH4 / 300 sccm H2 under ambient pressure for 30s yielded a graphene / copper system with approximately 60% surface coverage, predominantly single-layer, and island diameters of 1-5µm. Without any gold or carbon film treatment, the samples were directly cut into 3mm×3mm pieces, adhered to standard SEM aluminum posts with conductive adhesive, and surface particles were removed by blowing with an N2 gun.

[0034] 2. Equipment and Initial Calibration Instrument: Zeiss Sigma 360 field emission SEM. It should be noted that this technique is also applicable to other brands and models of SEM equipment. Equipped with an in-lens detector (…). Figure 1 TV mode (Scan Speed ​​= 16 frames / s) and Z-stage accuracy ≤ 1µm.

[0035] Emission current: 8.3µA (instrument default value); Astigmatism correction was performed on a 5µm×5µm area on copper foil under conditions of 5kV and WD=8mm, and saved as "Reference".

[0036] 3. Parameter Progression Strategy A two-step experimental method of "single-factor orthogonal" was used: 1) Single-factor experimental method: fix WD=4.5mm, V acc The voltage was gradually reduced from 1.0 kV to 0.1 kV to observe the contrast change and determine the optimal voltage range. 2) Orthogonal experimental method: Three levels of 0.5kV, 0.7kV and 1.0kV were selected near the optimal voltage, and three levels of WD of 0.5, 1.5 and 2.5mm were selected, for a total of 9 groups. The surface-subsurface mixing information index was recorded for each group.

[0037] 4. Imaging Process a) TV mode positioning: Magnification 2.00k×, find a suitable region and a region that contains two types of grains and has clear graphene island boundaries (clear grain boundaries can be observed in grain regions containing two crystal orientations), and mark it as ROI-1.

[0038] b) Pre-shoot under normal conditions: Switch to 5kV, WD=8mm, In-lens mode, pixel dwell time=1µs, 2048×1536 pixels, and take a "comparison image", such as... Figure 2 As shown.

[0039] c) Short WD setting: Return to TV mode and raise the sample stage in 0.5 or 1.0 mm increments until WD=4.5 mm.

[0040] d) Ultra-low voltage imaging: Maintain In-lens mode, and reduce V acc With a voltage of 1.0 kV, a probe current of 51 pA (beam spot ≈ 1.8 nm), a well time of 3 µs, and an integration frame count of 128, a high-contrast image of "1.0 kV - 4.5 mm" was obtained.

[0041] e) Cycle: Change V sequentially according to the combinations in Table 1. accWith WD, each time the WD is modified, the Z-axis approximation is first completed in TV mode before switching to In-lens shooting; the aperture and astigmatism are not adjusted throughout the process to ensure consistent electron beam parameters, such as... Figure 3 As shown.

[0042] 5. Key Controls and Data Logging Controllable electronic dose: The field of view can be uniformly set to 5µm × 3.75µm, and the dose per frame is 6.5e. - / nm 2 (Single-frame dose is not affected by the size of the field of view), total dose ≤830e - / nm 2 This prevents graphene from becoming charged or from accumulating contaminants.

[0043] Subsurface quantification: Employing a "surface-subsurface mixed information index". CV=( I - I 0) / I 0×100%, of which I 0 represents the grayscale value of the Cu substrate. I The grayscale value represents the area covered by graphene. A higher CV value indicates greater surface morphology contrast, which better meets the requirements for characterizing the nanoscale morphology of the sample's metasurface. The grayscale value refers to the brightness or darkness of a specific region on the sample surface.

[0044] Result: As Figure 3 As shown, the dashed box area represents the parameter range where the electron beam cannot be focused. The SEM image with good contrast in the yellow box area corresponds to acceleration voltage and working distance combinations of 0.3-0.7kV / 0.5-1.0mm and 0.5-1.0kV / 2.5-3.5mm. It is important to note that under ultra-low acceleration voltage and fixed working distance conditions, the contrast is best at 0.3-0.5kV. This is because the electron beam penetration depth is extremely small at ultra-low acceleration voltages, limited to the thickness of graphene on the copper substrate and part of the metasurface thickness of the copper substrate. According to the SE yield with increasing voltage... accThe electron beam penetration depth is the lowest at 0.1 kV, almost limited to the thickness of the graphene, but this also results in the worst image contrast and the lowest SE yield. At a working distance of 1.5 mm, backscattered electrons (BSE) generated by the interaction between the electron beam and the sample cannot generate type III secondary electrons (SE3) on the inner wall of the emission aperture; they can only be generated at the edge pole piece. The SE3 generated by impacting the pole piece cannot enter the detector (because the pole piece is located at the outer edge of the emission aperture). Therefore, the detector collects very little SE3 at this working distance. Combined with the attenuation effect of graphene, this makes the graphene-covered area darker than the substrate at this parameter. (Compared to 0.5 mm working distance, 1.5 mm reduces the effect of SE3, while the oxide layer has a greater influence on SE3 due to atomic number contrast).

[0045] In addition, since the 0.5-1.0mm working distance is more difficult to operate and can easily cause damage and contamination to the electron gun or sample, it is recommended to choose the 0.5-1.0kV / 2.5-3.5mm combination.

[0046] Figure 4 SEM images of Graphene / Cu samples under ultra-low accelerating voltage-short working distance and conventional accelerating voltage-working distance. Figure 4 As shown in Figure a, under ultra-low accelerating voltage and short working distance (Vacc=0.3kV, WD=0.5mm), the SEM images exhibit high morphological contrast, clearly revealing the graphene texture, i.e., the boundaries between graphene regions at different nucleation sites, thus realizing the nanoscale morphological features of the metasurface; Figure 4 As shown in b, when Vacc = 1.0 kV and WD = 3.5 mm, the graphene region shows relatively few visible textures, with only large folds or cracks visible; for example... Figure 4 As shown in Figure c, under the conditions of conventional accelerating voltage and working distance (Vacc=5.0kV, WD=7.1mm), the contrast between graphene and copper substrate is relatively small, and the detailed morphological features of the sample surface are relatively blurred.

[0047] Figure 5 The images show SEM images of grain regions with different crystal orientations in the Graphene / Cu sample of this invention under ultra-low accelerating voltage and short working distance. As can be seen from the figures, grain regions with different crystal orientations all exhibit nanoscale metasurface morphology contrast under ultra-low accelerating voltage and short working distance.

[0048] 7. Repeatability and Universality The above procedure was used to repeat the experiment on three batches of different copper-based graphene samples, and the relative standard deviation of the CV value was <6%; the method can be further applied directly to: —Materials field: Achromatic SiN superlens (step error < 2nm) —Catalysis field: Single-atom catalysts (support defects 5-8nm) —Biomedical field: Immobilized exosomes (membrane protein canopy thickness 3-5nm).

[0049] All samples achieved a vertical resolution of <1 nm and a surface contrast index of >80%, without the need for metal coating, and the electron dose was below the sample damage threshold.

[0050] in conclusion The above embodiments demonstrate that by precisely matching In-lens SE detection within the range of ultra-low accelerating voltage (≤1kV) and short working distance (≤5mm), the present invention successfully suppresses the electron injection depth, compresses the information collection range to within 1-2nm of the surface, and achieves direct visualization of the true morphology, sub-nanometer steps, and crystal orientation differences of metasurfaces, single-atom catalysts, and biological nanoparticles, without the need for any conductive coating. This solves the common problem of traditional SEM where surface signals are averaged by the subsurface layer and suffer severe charge damage at the "deep nano" scale.

[0051] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0052] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for characterizing nanoscale morphology contrast under ultra-low voltage and short working distance SEM, characterized in that, The method includes: S1: Place the sample on the SEM sample stage and select the target imaging area in TV mode; S2: Switch the SEM from TV mode to imaging mode, select the In-lens secondary electron detector, and perform astigmatism correction and focusing under normal accelerating voltage and working distance conditions; the normal accelerating voltage is greater than or equal to 5kV, and the working distance is greater than or equal to 8mm. S3: Switch the SEM from imaging mode back to TV mode, and shorten the working distance to the target short working distance while in visual mode; the target short working distance is less than 5mm. S4: Switch the SEM from TV mode back to imaging mode, select the In-lens secondary electron detector, reduce the accelerating voltage to an ultra-low accelerating voltage, and focus on the same target area; the ultra-low accelerating voltage is less than or equal to 1kV. Repeat steps S3 and S4 to perform system imaging under various parameter combinations of ultra-low accelerating voltage and short working distance, in order to obtain and compare nanoscale morphological contrast information of the sample metasurface.

2. The method as described in claim 1, characterized in that, The target imaging region is a grain containing two or more crystal orientations.

3. The method as described in claim 1, characterized in that, During system imaging by changing the accelerating voltage and / or working distance, the electron beam current and electron beam irradiation area remain constant.

4. The method as described in claim 1, characterized in that, The various parameter combinations of ultra-low acceleration voltage and short working distance are systematically screened using single-factor experimental methods, orthogonal experimental methods, or a combination of both.

5. The method as described in claim 1, characterized in that, The ultra-low acceleration voltage is 0.1kV to 1.0kV.

6. The method as described in claim 1 or 5, characterized in that, The short working distance is 0.5mm to 5.0mm.

7. The method as described in claim 1, characterized in that, The optimized combination of ultra-low acceleration voltage and short operating distance includes: A combination of 0.3kV-0.7kV and 0.5mm-1.0mm; or a combination of 0.5kV-1.0kV and 2.5mm-3.5mm.

8. The method as described in claim 3, characterized in that, The electron beam current is fixed at 51 pA, and the single-frame electron dose is 6.5 e. - / nm 2 The total dose shall not exceed 830e - / nm 2 .

9. The method as described in claim 1, characterized in that, The sample is a conductive or semi-conductive material or nanoparticle that does not require metal sputtering, including graphene, metasurface materials, single-atom catalysts, or biological tissue samples, used to achieve metasurface nanoscale morphology characterization of the sample.

10. The method as described in claim 1, characterized in that, The method further includes performing contrast quantitative analysis on the acquired SEM images to calculate the surface-subsurface mixing information index (CV), the formula of which is: CV=( I - I 0) / I 0×100%, in, I 0 represents the average grayscale value of the substrate region. I This represents the average grayscale value of the image for the surface feature region.

Images