A method of microanalysis of liquid environmental samples of in-situ thick liquid layers by scanning electron microscopy

Abstract

The present application relates to a kind of scanning electron microscope in-situ thick liquid layer of liquid environment sample of microanalysis method, it includes the following steps: S1, liquid environment sample is sealed in sealed film and is sealed to obtain assembly sample;S2, the micro area characterization test of thick liquid layer of liquid environment sample is carried out in field emission scanning electron microscope cavity, the difference of the image contrast caused by the reflection ability of sample and liquid environment to backscattered electron beam is different, obtain the high resolution topographic map of liquid environment sample;S3, the element analysis of micro area of liquid environment sample is carried out using the characteristic X-ray energy dispersive spectrometer equipped with scanning electron microscope, accurately obtain the element composition and element distribution information of liquid environment sample.According to the microanalysis method of scanning electron microscope in-situ thick liquid layer of liquid environment sample of the present application, the backscattered electron beam of high resolution field emission scanning electron microscope equipped with energy dispersive spectrometer and the in-situ observation of the micro area morphology and spectroscopy of thick liquid layer of liquid environment sample of energy spectrum characterization are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to a characterization method of in-situ electron microscope observation samples in liquid environment, and more particularly to a microanalysis method of in-situ thick liquid layer liquid environment samples of a scanning electron microscope. BACKGROUND

[0002] Optical microscopes are known for observation of solid samples, but they only have a thousand times magnification, and the transmission electron microscope observation field is too small, about one hundred microns. Compared with the insufficient resolution of optical microscopes and the too small observation field of transmission electron microscopes, field emission scanning electron microscopes have become valuable research tools with the advantages of optical microscopes and transmission electron microscopes, with their nanometer-level high spatial resolution, large millimeter-level observation field range, second-level fast imaging, and multi-element full-spectrum analysis capabilities.

[0003] However, for liquid environment samples, especially for liquid environment samples with a liquid layer thickness of more than one hundred nanometers to microns, field emission scanning electron microscopes face serious challenges and difficulties. First, the high vacuum environment of the scanning electron microscope will cause the liquid to evaporate in the vacuum, affecting the vacuum degree and contaminating the electron microscope cavity. Second, the usual secondary electron imaging observation mode of the scanning electron microscope will be scattered by the liquid layer, resulting in extremely low resolution and making it difficult to capture the high-resolution morphology of the sample in the liquid. In addition, the secondary electron beam of the scanning electron microscope has an incident depth of only about 10 nm, which can only detect surface information of the sample, and it is difficult to detect sample information in the liquid layer with a depth of more than one hundred nanometers to microns.

[0004] With the development of chip technology, scanning electron microscope liquid environment samples can be sealed in a thin film to avoid evaporation in a high vacuum environment, thereby affecting the electron microscope vacuum degree and contaminating the electron microscope cavity. However, the low penetration depth of secondary electrons and the multiple scattering of the electron beam by the liquid still severely limit the application of scanning electron microscopes in observing liquid environment samples.

[0005] Although a commercial scanning electron microscope transmission imaging observation mode has been developed, it not only requires the purchase of expensive scanning transmission electron probes, special sample fixing brackets and other equipment, but also requires modification of the electron microscope, and further requires that the liquid layer of the liquid environment sample be very thin, with a thickness of only a few tens of nanometers to one hundred nanometers, as described in CN201510315422.2. This makes it impossible to observe some relatively thick liquid environment samples, such as micron-sized cells, micron-sized bubbles, and one hundred nanometer-sized particles, etc. Further, it affects the development of the fields of biomedicine, energy environment, and micro-nano technology, etc. SUMMARY

[0006] In order to solve the technical difficulties in the prior art that the scanning electron microscope detects the liquid environment sample of the thick liquid layer, the application provides a microscopic analysis method for in-situ thick liquid layer of liquid environment sample of a scanning electron microscope, which replaces the traditional scanning electron microscope secondary electron beam imaging mode with a backscattered electron beam imaging mode, and cooperates with a characteristic X-ray spectrometer to detect the morphology and composition of the liquid environment sample with a thickness of more than 100 nanometers to microns.

[0007] The microscopic analysis method for in-situ thick liquid layer of liquid environment sample of a scanning electron microscope according to the application comprises the following steps: S1, encapsulating the liquid environment sample in a sealed film to obtain an assembled sample; S2, performing micro-area characterization testing on the thick liquid layer of the liquid environment sample in a field emission scanning electron microscope cavity, and obtaining a high-resolution morphology map of the liquid environment sample by using the difference in image contrast caused by the different reflection abilities of the sample and the liquid environment to backscattered electron beams; and S3, performing element analysis on a microscopic area of the liquid environment sample by using a characteristic X-ray spectrometer equipped with the scanning electron microscope, and accurately obtaining element composition and element distribution information of the liquid environment sample.

[0008] Preferably, the thickness of the liquid environment sample is 100 nm to 2 microns.

[0009] Preferably, a backscattered electron probe is inserted into the field emission scanning electron microscope cavity to collect backscattered electrons reflected from the surface of the sample in the liquid environment, so as to obtain micro-nano high-resolution morphology information of the sample.

[0010] Preferably, an objective lens diaphragm is inserted into the field emission scanning electron microscope cavity, and the size of the objective lens diaphragm is between 20 microns and 40 microns.

[0011] Preferably, the working distance between the sample and the electron gun in the field emission scanning electron microscope cavity is between 7 mm and 9 mm.

[0012] Preferably, an EDS spectrometer probe is inserted into the field emission scanning electron microscope cavity to obtain characteristic X-rays emitted by the sample and obtain element distribution information of the sample.

[0013] Preferably, the sealed film is a silicon nitride film, a multilayer carbon film, a graphene film or an oxidized graphene film.

[0014] Preferably, the liquid environment of the liquid environment sample is water, liquid alcohol, liquid ketone or liquid phenol.

[0015] Preferably, the sample of the liquid environment sample is an animal or plant cell, bacteria, a micro-nano particle, a micro-nano suspension droplet or a micro-nano bubble.

[0016] Preferably, the resolution of the high-resolution field emission scanning electron microscope in characterizing the liquid environment sample is 50 nm to 1 micron.

[0017] The microanalysis method for in-situ thick liquid layer liquid environment sample of scanning electron microscope according to the application realizes the in-situ observation of micro-area morphology and spectroscopy of the thick liquid layer liquid environment sample by the backscattered electron beam and energy spectrum of the high-resolution field emission scanning electron microscope equipped with an energy spectrometer. In summary, the application provides a simple, efficient and fast method for microimaging and spectroscopy detection of the thick liquid layer liquid environment sample of scanning electron microscope, which realizes the accurate characterization of micro-area morphology and spectroscopy of the liquid sample by the backscattered electron beam and energy spectrum of the high-resolution field emission scanning electron microscope equipped with an energy spectrometer, overcomes the defects of low resolution and inability of high-resolution imaging of the liquid environment sample when the liquid layer thickness exceeds hundreds of nanometers, and overcomes the deficiency of the need for special purchase of expensive scanning transmission sample holder and detector and modification of the electron microscope for the observation of in-situ liquid environment sample by scanning electron microscope through reasonable adjustment of imaging parameters and matching of energy spectrum. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a schematic diagram of the microanalysis method for in-situ thick liquid layer liquid environment sample of scanning electron microscope according to the application.

[0019] Figure 2 is a morphology diagram of oxygen micro-bubbles in water environment according to example 1 of the application.

[0020] Figure 3 is Figure 2 the oxygen element distribution diagram in.

[0021] Figure 4 is a morphology diagram of cells in water environment according to example 3 of the application.

[0022] Figure 5 is Figure 4 the sodium element distribution diagram in.

[0023] Figure 6 is Figure 4 the oxygen element distribution diagram in. DETAILED DESCRIPTION

[0024] The preferred embodiments of the application are described in detail below with reference to the accompanying drawings.

[0025] The key of the present application is to characterize the liquid phase by field emission scanning electron microscopy. It should be understood that the scanning electron microscopy application technology for in-situ sample of liquid environment with liquid layer thickness exceeding 100 nm is also very difficult in the prior art. This is mainly due to the very low electron beam penetration of field emission scanning electron microscopy, especially for liquid environment samples with thickness exceeding 100 nm. The secondary electron detection depth of the secondary electron imaging observation mode of the conventional field emission scanning electron microscopy is only about 10 nm, which can only detect the surface information of the sample, and will be scattered by the liquid layer multiple times, resulting in inability to image and difficulty in capturing the sample and distinguishing the sample from the liquid environment impurities, liquid phase undissolved reactants or chip defects.

[0026] Referring to Figure 1 The microanalysis method of the in-situ thick liquid layer liquid environment sample of the scanning electron microscope according to the present application first includes encapsulating the liquid environment sample 1 in the silicon nitride film 2, and sealing the assembled sample with a vacuum ester. The liquid environment of the liquid environment sample 1 includes but is not limited to water, alcohol, ketone, phenol and the like. The sample 7 in the liquid environment sample 1 includes but is not limited to animal and plant cells, bacteria, micro-nano particles, micro-nano suspension droplets, micro-nano bubbles and the like. The thickness of the liquid environment sample 1 is 100 nm to 5 μm. Preferably, the thickness of the liquid environment sample 1 is 100 nm to 1 μm. The thickness of the silicon nitride film 2 is 50 to 100 nm.

[0027] The microanalysis method of the in-situ thick liquid layer liquid environment sample of the scanning electron microscope according to the present application next includes testing the liquid environment sample 1 in the field emission scanning electron microscope cavity 3, utilizing the difference in image contrast caused by the different reflection abilities of the sample 7 and the liquid environment to the electron beam, forming a high-resolution image to characterize the sample; and then utilizing the EDS spectrometer probe 4 equipped with the scanning electron microscope to analyze the elements in the micro area of the liquid environment sample 1, accurately characterizing the element information of the sample.

[0028] The high-resolution field emission scanning electron microscope has the characteristics of nanoscale rapid imaging and rapid multi-element spectrum analysis, and can rapidly and accurately characterize and analyze the liquid environment sample 1 by simultaneously observing the morphology and composition information of the sample. This is mainly because the high-resolution field emission scanning electron microscope has a large observation field range, rapid imaging and multi-element spectrum analysis capability, and is a valuable research means combining the advantages of optical microscopes (similar to the liquid cell size that can be observed in optical microscopes) and transmission electron microscopes (high resolution, up to several nanometers). Moreover, the scanning electron microscope imaging and element analysis speed of the high-resolution field emission scanning electron microscope is fast, and the test results can be obtained quickly, which is very suitable for in-situ observation of the changes of the sample 7 in the liquid environment.

[0029] Specifically, the assembled sample is transferred to the sample stage 5, and then the sample stage 5 is placed in the field emission scanning electron microscope cavity 3, and vacuum is drawn. When the vacuum degree reaches 2.5 × 10 -5When the pressure in the chamber is 10~10 mbar, the electron beam of the scanning electron microscope is turned on, the voltage of the electron beam is continuously adjustable between 5~30 kV, the objective aperture is selected, the size of the objective aperture is in the range of 10~120 μm, the working distance of the sample stage 5 is adjusted, the working distance of the sample stage 5 is continuously adjustable between 2~15 mm, the backscattered electron probe 6 is inserted, and the backscattered electron signal is received, and the EDS spectrometer probe 4 is inserted to detect the element distribution information of the sample.

[0030] The objective aperture is used to select the scanning observation range, and the size is preferably between 20~40 μm. In the preferred embodiment, the size of the objective aperture is 30 μm, which is higher than the backscattered image resolution of the conventional 60 μm objective aperture used for backscattered imaging.

[0031] The working distance between the sample and the electron gun is used to ensure the resolution of the image, and the distance is preferably between 7~9 mm (for example, 8.5 mm). In this way, the best backscattered imaging and element characterization of the spectrum can be achieved at the same time.

[0032] The imaging mode of the high-resolution field emission scanning electron microscope is backscattered electron imaging (BSE), which can quickly analyze various different phase compositions in the sample. The backscattered electron probe 6 is used to collect the backscattered electrons reflected from the surface of the liquid environment sample 1 to obtain the micro-nano morphology information of the sample 7. The present application uses the backscattered electron beam to image the thick liquid layer liquid environment sample by reasonably selecting the size of the objective aperture and the working distance, which can overcome the shortcomings of poor penetration of secondary electrons and inability to image in a liquid environment, and can overcome the shortcomings of the use of a scanning transmission electron beam, the need to purchase an expensive special transmission electron probe and a sample holder, the need to modify the electron microscope, and the need for extremely thin liquid sample thickness. The backscattered electron beam used in the present application has strong penetration in liquid, and although it is a reflection light path imaging, it can penetrate a micron-thick liquid layer. At the same time, by selecting an objective aperture with a size of 20~40 μm, the imaging resolution can be effectively improved without the need for additional detectors, sample holders, and modifications to the electron microscope, and without the need for harsh restrictions on the thickness of the sample liquid layer.

[0033] The resolution of the backscattered electron beam of the high-resolution field emission scanning electron microscope in characterizing the liquid environment sample 1 is 50 nm~1 μm. By selecting an objective aperture with a size of 20~40 μm (for example, 30 μm), the resolution of the backscattered electron beam probe will be affected and decreased in a micron-thick liquid layer, but it can detect a micron-thick liquid layer, thereby widening the characterization range of the liquid environment sample.

[0034] The voltage range of the high resolution field emission scanning electron microscope is 5-30 kV. It should be understood that below 5 kV will make the signal insufficient for clear imaging, and above 30 kV will exceed the limit voltage value of the field emission electron microscope. The preferred voltage range of the high resolution field emission scanning electron microscope is 15-25 kV, for example 20 kV.

[0035] The EDS energy spectrum probe 4 is used to obtain characteristic X-rays emitted by the liquid environment sample 1, to perform rapid element distribution analysis, and to accurately obtain the element composition and element distribution information of the sample 7 in spectroscopy.

[0036] Thus, the present application proposes a method for characterizing a liquid environment sample of a thick liquid layer in situ by a scanning electron microscope, which is simple, rapid and accurate, and which is capable of characterizing a liquid environment sample with a thickness exceeding 100 nm by using a high spatial resolution scanning electron microscope backscattered electron beam imaging mode. Specifically, the high resolution field emission scanning electron microscope is used to characterize the micro-nano morphology and element analysis of the liquid environment sample 1 of the thick liquid layer, so as to obtain the high resolution morphology and element composition and distribution information of the sample 7 in the liquid environment.

[0037] The present application provides a simple, efficient and accurate method for characterizing a liquid environment sample of a thick liquid layer in situ by a scanning electron microscope. The backscattered electron beam and energy spectrum characterization of the high resolution field emission scanning electron microscope equipped with an energy spectrum analyzer are used to accurately perform microscopic analysis of the morphology and spectroscopy of the sample 7, which makes up for the shortcomings of the shallow secondary electron beam incident depth of the scanning electron microscope, the difficulty in imaging in the liquid environment, and the need to rely on the purchase of expensive commercial scanning transmission sample holders and probes and the modification of the electron microscope, and overcomes the defect that the traditional scanning electron microscope cannot perform high resolution imaging on the liquid environment sample with a liquid layer thickness exceeding 100 nm.

[0038] A Zeiss Gemini 300 field emission scanning electron microscope is used to image and analyze the scanning transmission microscopic liquid sample of the soft X-ray packaged by the silicon nitride window. The observation conditions are as follows: acceleration voltage: 5-30 kV, objective aperture: 10-120 μm, working distance: 2-15 mm, magnification: 50-1000000X, and sample liquid layer thickness: 100 nm-2 μm.

[0039] Example 1

[0040] The oxygen micron bubbles in the water liquid environment can be imaged by backscattered electrons, and the element analysis by the energy spectrum analyzer can be used to determine the morphology and element distribution information of the oxygen micron bubbles, as shown in Figure 2 and Figure 3 .

[0041] Example 2

[0042] Carbon fluoride micron suspension droplets in water environment can be imaged by backscattered electrons, and element analysis can be performed by an energy spectrometer to obtain the morphology and element distribution information of the carbon fluoride micron suspension.

[0043] Embodiment 3

[0044] Cell samples in water environment can be imaged by backscattered electrons, and element analysis can be performed by an energy spectrometer to obtain the morphology and element distribution information of the cells, as shown in Figures 4-6 .

[0045] The characteristic X-ray energy spectrometer equipped by the high-resolution field emission scanning electron microscope can simultaneously analyze elements 5 (B) to 98 (Cf) in the periodic table, and can perform multi-element rapid full-spectrum analysis on the sample.

[0046] The above is only a preferred embodiment of the present application, and is not intended to limit the scope of the present application. The above embodiment of the present application can also be variously changed. Any simple, equivalent changes and modifications made in accordance with the content of the claims and the specification of the present application fall within the scope of the claims of the present patent. The present application is not described in detail, and is conventional technical content.

Claims

1. A method of microanalysis of a liquid environmental sample of a thick liquid layer in situ in a scanning electron microscope, characterized in that, The microscopic analysis method comprises the following steps: S1, encapsulating a liquid environment sample with a thickness of 100 nm to 2 µm in a sealed film to obtain an assembled sample, the sample of the liquid environment sample being animal and plant cells, bacteria, micro-nano particles, micro-nano suspension droplets or micro-nano bubbles; S2, in the field emission scanning electron microscope cavity, inserting an objective diaphragm with a size of 20-40 µm, performing micro-area characterization test on the thick liquid layer of the liquid environment sample, and obtaining high-resolution morphology of the liquid environment sample by using the difference in image contrast caused by the different reflection abilities of the sample and the liquid environment to backscattered electron beams; S3, using the characteristic X-ray energy spectrometer equipped with the scanning electron microscope to perform element analysis on the micro area of the liquid environment sample, and accurately obtaining the element composition and element distribution information of the liquid environment sample.

2. The microscopic analysis method according to claim 1, characterized by, A backscattered electron probe is inserted into the field emission scanning electron microscope cavity to collect backscattered electrons reflected from the surface of the sample in the liquid environment to obtain micro-nano high-resolution morphology information of the sample.

3. The microscopic analysis method according to claim 1, characterized by, In the field emission scanning electron microscope cavity, the working distance between the sample and the electron gun is between 7-9 mm.

4. The method of microscopic analysis according to claim 1, wherein, An EDS energy spectrometer probe is inserted into the field emission scanning electron microscope cavity to obtain characteristic X-rays emitted by the sample and obtain element distribution information of the sample.

5. The method of microscopic analysis according to claim 1, wherein, The sealing film is a silicon nitride film, a multi-layer carbon film, a graphene film or an oxidized graphene film.

6. The method of microscopic analysis according to claim 1, wherein, The liquid environment of the liquid environment sample is water, liquid alcohol, liquid ketone or liquid phenol.

7. The method of microscopic analysis according to claim 1, wherein, The resolution of the high-resolution field emission scanning electron microscope in characterizing the liquid environment sample is 50 nm-1 µm.

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