Specimen preparation for transmission electron microscopy

1. Technical Field

The present invention relates to preparation of a specimen suitable for observation under a transmission electron microscope (TEM), which reflects the true distribution status of nanoparticles in a blood sample, either in dispersion or agglomeration.

2. Description of Related Art

FIG. 1A-E show a first specimen prepared according to a traditional method.

FIG. 1A shows a traditional substrate 10 , which is usually a piece of copper.

FIG. 1B shows a drop of blood sample 11 placed on the top surface of the substrate 10 . The blood sample 11 contains, among others, nanoparticles 11 N, and blood cells 11 C. The average diameter of a red blood cell (RBC) is 6 to 8 micrometers. The average diameter of a white blood cell (WBC) is 10 to 12 micrometers. A blood cell 11 C has a dimension in μm, which is larger than the dimension of a nanoparticle.

FIG. 1C shows a drop of blood sample 11 evaporates during drying. The drop of blood sample shrinks and a plurality of smaller droplets are formed. The surface tension 13 of each droplet drags the components therein closer and closer. The components undergo gatherings.

FIG. 1D illustrates two groups of aggregates A1 of nanoparticles 11 N are formed. The concentrating effect of the components within each droplet is caused by a surface tension 13 during drying, causing formation of aggregates A1 of nanoparticles. The aggregates A1 of nanoparticles in the prepared sample had appearances similar to nanoparticle-agglomerates, which may cause a confusion between aggregates and agglomerates and give wrong information to an observer when observing under TEM.

FIG. 1E shows a top view of FIG. 1D . Two groups of aggregates A1 of nanoparticles 11 N are formed. The specimen of FIG. 1E does not reflect the true status of nanoparticles 11 N, which is evenly dispersed in the blood sample 11 as evidenced by FIG. 1B .

Now, paying attention to nanoparticles 11 N only. One of the purposes to examine a specimen of blood sample is to observe the status of nanoparticles 11 N in the original blood sample, either in dispersion or agglomeration. However, a specimen prepared by a traditional method does not reflect the original or true status of nanoparticles 11 N in the original blood sample, either in dispersion or agglomeration. As shown in FIGS. 1D-E , which shows aggregates A1 of nanoparticles 11 N, false information has been displayed under TEM due to a surface tension 13 between the droplets during drying. It is desired that the true status of nanoparticles 11 N in the original blood sample can be reflected, either in dispersion or agglomeration.

FIGS. 2A-E show a second specimen prepared according to a traditional method.

FIG. 2A shows a traditional substrate 10 , a copper grid.

FIG. 2B shows a drop of blood sample 11 placed on the top surface of the substrate 10 . The blood sample 11 contains, among others, dispersed nanoparticles 11 N, nanoparticle-agglomerates 11 NA, and blood cells 11 C.

FIG. 2C shows the liquid evaporates during drying, the drop of blood sample shrinks and smaller droplets are formed. The surface tension 13 of each droplet drags the components closer and closer.

FIG. 2D shows aggregates A2 of nanoparticles are formed.

FIG. 2E shows the top view of FIG. 2D . Two groups of aggregates A2 of nanoparticles 11 N are formed. Actually, the aggregates A2 do not exist in the original blood sample, see FIG. 2B . The specimen of FIG. 2E gives false information to an observer.

Now, paying attention to nanoparticle-agglomerates 11 NA only. One of the purposes to examine a specimen of a blood sample under TEM is to observe whether any nanoparticle-agglomerate exists in an original blood sample. However, a specimen prepared by a traditional method does not reflect the true number of nanoparticle-agglomerates 11 NA. Several aggregates A2 of nanoparticles 11 N, counterfeits of nanoparticle-agglomerates 11 NA, are present in FIGS. 2D-E . The aggregates A2 are caused by the surface tension 13 of the droplets during drying. It is desired that the true situation of nanoparticles in the specimen can be observed, either in dispersion or agglomeration.

FIGS. 1A-E show the first specimen prepared according to a traditional method.

FIGS. 2A-E show the second specimen prepared according to a traditional method.

FIGS. 3A-E show the first specimen prepared according to the present invention.

FIGS. 4A-E show the second specimen prepared according to the present invention.

FIG. 5 is a perspective view of a specimen kit.

FIG. 6 is a section view of the first specimen kit according to the present invention.

FIGS. 7A-B illustrate the top substrate 20 T and the chamber of the first specimen kit, respectively.

FIGS. 8A-B illustrate the top substrate 30 T and the chamber of the second specimen kit, respectively.

FIGS. 9A-B illustrate the top substrate 40 T and the chamber of the second specimen kit, respectively.

This invention discloses a method of preparing a specimen with a specimen kit which has a tiny chamber. The height of the chamber is configured to be smaller than the diameter of a red blood cell (RBC). A RBC is smaller than a white blood cell (WBC). Thus, all blood cells are screened from entering the chamber of the specimen kit. The absence of blood cells in the specimen reduces interference with observation of nanoparticles, and therefore enhances the quality and quantity check of the specimen. The small chamber of the specimen kit limits a blood sample inside and eliminates the effect of surface tensions during drying. The specimen prepared according to the invention makes it possible to detect the true distribution status of nanoparticles in the original blood sample, either in dispersion and/or agglomeration.

FIGS. 3A-E illustrate the first specimen prepared according to the present invention.

FIG. 3A shows a blood sample 11 is ready to be injected into a chamber 20 of a specimen kit. The blood sample 11 contains nanoparticles 11 N and blood cells 11 C. A chamber 20 is formed between a top substrate 20 T and a bottom substrate 20 B. The height h between the top substrate 20 T and the bottom substrate 20 B is less than the diameter of a red blood cell (RBC). Thus, all RBCs and white blood cells (WBCs) are screened from entering the chamber 20 . A height of 10 μm for the chamber 20 is enough for TEM observation of the distribution status of nanoparticles in the blood sample, either in dispersion or agglomeration.

FIG. 3B shows nanoparticles 11 N in a blood sample 11 entering the chamber 20 . Blood cells 11 C do not enter the chamber 20 due to their larger dimensions.

FIG. 3C shows a drying process is performed to the blood sample 11 of- FIG. 3B . The blood sample 11 within the chamber 20 is dried and a plurality of small droplets are formed. Each droplet wraps a single nanoparticle 11 N and attaches onto the inner surface of the chamber 20 due to the adhesion force 13 of the droplet.

FIG. 3D shows some nanoparticles 11 N attached to the bottom surface of the top substrate 20 T after drying, and some nanoparticles 11 N attached to the top surface of the bottom substrate 20 B after drying. The tiny height h between the two substrates 20 T, 20 B limits the number of the nanoparticles 11 N distributed in Z direction and hence, eliminates the possibility of aggregation of the nanoparticles 11 N in Z direction.

FIG. 3E is a top view of FIG. 3D . Dispersed nanoparticles 11 N shown in FIG. 3E display the real dispersion status of nanoparticles 11 N in the original blood sample 11 as shown in FIG. 3B .

FIGS. 4A-E illustrate the second specimen prepared according to the present invention.

FIG. 4A shows a blood sample 11 ready to be injected into a chamber 20 of a specimen kit. The blood sample 11 contains nanoparticle-agglomerates 11 NA, nanoparticles 11 N, and blood cells 11 C. The chamber 20 is formed between a top substrate 20 T and a bottom substrate 20 B. The height h between the top substrate 20 T and the bottom substrate 20 B is less than the diameter of an RBC. Thus, all blood cells 11 C are screened from entering the chamber 20 .

FIG. 4B shows both nanoparticles 11 N and nanoparticle-agglomerates 11 NA in a blood sample 11 entering the chamber 20 . Blood cells 11 C do not enter the chamber 20 due to their larger dimensions.

FIG. 4C shows a drying process is performed to the blood sample 11 of FIG. 4B . The blood sample 11 within the chamber 20 is dried and a plurality of small droplets are formed. Each droplet wraps a single nanoparticle and attaches onto the inner surface of the chamber 20 due to the adhesion force 13 of the droplet.

FIG. 4D shows some nanoparticles 11 N and nanoparticle-agglomerates 11 NA attached to the bottom surface of the top substrate 20 T, and some nanoparticles 11 N and nanoparticle-agglomerates 11 NA attached to the top surface of the bottom substrate 20 B after drying. The tiny height h limits the numbers of nanoparticles 11 N and nanoparticle-agglomerates 11 NA distributed especially in Z direction, and hence eliminates the possibility of aggregation of nanoparticles 11 N and nanoparticle-agglomeration 11 NA in Z direction.

FIG. 4E shows the top view of FIG. 4D . The dispersed nanoparticles 11 N and nanoparticle-agglomerates 11 NA in the specimen display the real situation of nanoparticles 11 N and nanoparticle-agglomerates 11 NA distributed in the original blood sample 11 as shown in FIG. 4B .

FIG. 5 is a perspective view of a specimen kit.

FIG. 5 shows a specimen kit suitable for preparing a specimen for observation under a TEM. The kit has a chamber 20 formed between a top substrate 20 T and a bottom substrate 20 B. The height h of the chamber 20 is smaller than the diameter of an RBC, and the top substrate 20 T is made of a material transparent to electrons. A chamber height of 10 μm is enough for TEM observation of the distribution status of nanoparticles in a blood sample 11 , either in dispersion or agglomeration. A spacer 22 is inserted between the substrates to control the height. A solution entrance 26 is configured for injection of a sample. Observation window 25 is made at the center and on the top of a frame 24 of the kit. Part of the chamber 20 is exposed to the window 25 for TEM observation from the top of the kit.

FIG. 6 is a section view of the first specimen kit according to the present invention.

FIG. 6 shows a chamber 20 formed between a top substrate 20 T and a bottom substrate 20 B. An observation window 25 is made at the center and on the top of a frame 24 of the kit. Part of the chamber 20 is exposed to the window 25 for TEM observation from the top of the kit. A solution entrance 26 is configured for sample injection.

FIGS. 7A-B illustrate the top substrate 20 T and the chamber of the first specimen kit.

FIG. 7A shows the top view of the chamber 20 of the first specimen kit. The top substrate 20 T is a flat panel transparent to electrons. FIG. 7B is a section view of FIG. 7A , showing the top substrate 20 T and the bottom substrate 20 B, and a blood sample 11 filled in the chamber between the substrates 20 T, 20 B.

FIGS. 8A-B illustrate the top substrate 30 T and the chamber of the second specimen kit.

FIG. 8A is a top view of the second specimen kit, showing the top substrate 30 T and a plurality of through holes 31 made in the top substrate 30 T. When a specimen with blood or liquid is present in the chamber, the holes 31 make observation of the specimen easier.

FIG. 8B is a section view of FIG. 8A , showing the top substrate 30 T, the bottom substrate 30 B, and a blood sample 11 filled in the chamber between the substrates 30 T, 30 B. Each hole is configured to be small enough to keep the blood sample 11 stay in the chamber due to a surface tension 33 . Thus, the blood sample 11 does not seep through the holes 31 .

FIGS. 9A-B show the top substrate 40 T and the chamber of the second specimen kit.

FIG. 9A is a top view of the third specimen kit, showing the top substrate 40 T, and a plurality of through grooves 41 made in the top substrate 40 T. When a specimen with blood or liquid is present in the chamber, the grooves 41 made observation of the specimen easier.

FIG. 9B is a section view of FIG. 9A , showing the top substrate 40 T, the bottom substrate 40 B, and a blood sample 11 filled in the chamber between the substrates 40 T, 40 B. Each through groove 41 is configured to be small enough to keep the blood sample 11 stay in the chamber due to a surface tension 43 . Thus, the blood sample 11 does not seep through the grooves 41 .

While several embodiments have been described by way of examples, it will be apparent to those skilled in the an that various modifications may be configured without departing from the spirit of the present invention. Such modifications are all within the scope of the present invention, as defined by the appended claims.