Abstract:
A specimen kit having a tiny chamber is disclosed for a specimen preparation for TEM. The space height of the chamber is far smaller than dimensions of blood cells and therefore is adapted to sort nanoparticles from the blood cells. The specimen prepared under this invention is suitable for TEM observation over a true distribution status of nanoparticles in blood. The extremely tiny space height in Z direction eliminates the possibility of aggregation of the nanoparticles and/or agglomeration in Z direction during drying; therefore, a specimen prepared under this invention is suitable for TEM observation over the dispersion and/or agglomeration of nanoparticles in a blood.

Description:
BACKGROUND 
     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. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         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. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     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.