SCANNING ELECTRON MICROSCOPE

The invention relates to an electrically con- ducting electrode for analysis of multiple samples in a scanning electron microscope.

It is known per se to arrange the electrode as a sample holder with a surface (analysis surface) for supporting the sample. In that case the sample may be collected and enriched on the sample holder and can be analyzed in the sample chamber of the scanning electron microscope without it being necessary to move the sample between several sample holders. By the surface supporting the sample being constructed as a filter, possibly having two or more demountable porous surfaces of different pore sizes, the electrode can be used for collecting particles, if any, of different sizes, entrained in the sample material, on different surfaces and for enriching these particles by a filtration procedure, but it is also possible to make the surface supporting the sample impervious and to collect and enrich the sample material by centrifugal force or by an electromagnetic field. By means of the filtration method small par¬ ticles such as virus, bacteria, fungus, pollen, and asbestos fibres, entrained in liquid or gas, can be applied directly to the support surface/filter sur¬ face by the gas or the liquid, respectively, being sucked through the filter disc. When a particle sample has been applied to the support surface of the sample holder, the sample and also the sample holder - at least on the upper side thereof - is coated with a very thin (5 Angstrom - 500 Angstrom) layer which is applied by evaporation after evacu-

ation of all air in a vacuum chamber. This layer as well as the sample holder is electrically con¬ ducting, and therefore the sample holder can then be used directly as an electrode in a scanning electron microscope for investigation of the collected par¬ ticle sample without moving the collected particles from one place to another.

After repeated air evacuation in a vacuum cham¬ ber analysis of particles collected from air or liquid, e.g. from body liquid, can be effected in the scanning electron microscope. The result of the analysis made in this way can be of critical import¬ ance for taking specific measures, e.g. for control, decontamination, or terapeutical treatment, for which it is necessary that the result of the analy¬ sis is absolutely reliable; in other words, no con¬ fusion of samples from different places or different persons should be possible because the consequences thereof may be disastrous. At the same time there may be a demand for a rational handling of several samples simultaneously without opening the sample door in the scanning electron microscope such that the analyses can be made quickly and comparative analyses can be performed. By means of computerized picture analysis a great number of samples then can be analyzed automatically without human efforts. At present only one sample at a time can be analyzed in the sample chamber of the- scanning electron microscope because it has not been possible so far to identify in a rational manner the individ¬ ual samples if several samples are inserted simul¬ taneously in the sample chamber. Since a confusion of samples which are being investigated simulta¬ neously may be disastrous, as mentioned above it has therefore been necessary to adhere to individual in-

vestigation of the individual samples by inserting these samples one at a time into the sample chamber of the scanning electron microscope, which involves a long time of analysis considering the fact that each air evacuation takes from 5 to 30 minutes.

In order to make possible that several samples are introduced simultaneously into the sample chamber of the scanning electron microscope for analysis while saving considerable time without any risk of confusion of the samples the invention pro¬ vides an electrode of the kind referred to above having the characteristics of claim 1.

In order to explain the invention in detail reference is made to the accompanying drawing in which

FIG. 1 is a partly exploded view of an elec¬ trode of the invention,

FIG. 2 is a vertical sectional view of the electrode in FIG. 1, FIG. 3 is an enlarged vertical sectional view of an individual sample holder,

FIG. 4 is a plan view of the sample holder in FIG. 3, and

FIG. 5 is a perspective view showing an alter- native embodiment of the electrode.

In the drawing FIGS. 1 and 2 disclose an elec¬ trode which in this case comprises an annular re¬ volving disc 10 having a central hub 11. This hub has a guide slot 12 to engage a corresponding guide rib on a pivot in a scanning electron microscope.

The revolving disc thus can be mounted to the pivot only in a definite relative rotational position, and if the pivot is then connected to an indexing device it is possible to determine exactly the rotational position of the electrode in the scanning electron

microscope. On the upper side of the revolving disc there are provided eight pins 13. However, the re¬ volving disc can be arranged with a varying number of pins which always should be uniformly spaced such that when the samples are automatically run through in the sample chamber of the scanning electron microscope one will always land on the surface of the next sample holder. The revolving disc proper is not necessarily annular; the disc may have square, rectangular, or elliptical shape. The pins 13 are slightly conical and may be constructed as the male element of a Luer coupling. Each pin has a through passage 14 which communicates with the hub through a passage 15 in the revolving disc. On each pin a sample holder 16 is detachably mounted, said holder in this case being constructed as disclosed in WO 86/02160. The holder according to FIGS. 3 and 4 comprises a base portion 17 which forms a cavity 18 communicating through the bottom thereof with a hollow stud 19. This stud forms the female element of a Luer coupling so as to be de¬ tachably mounted to one of the pins 13. A filter disc 20 forms a supporting surface for the sample and is secured and sealed at the periphery thereof between an abutment surface surrounding the cavity, and a lock ring 21 attached to the base portion, said lock ring engaging the upper side of the filter disc at an annular flange 22. In the hollow stud 19 a groove 23 is provided to receive a projection 24 on the pin such that the sample holder can be mounted in a single predetermined rotational posi¬ tion only on the pin.

On the lock ring 21 at the surface supporting the sample on the filter disc 20, viz. on the circu- lar annular surface around the filter disc, formed

by the lock ring 21, a code 25 formed by a struc¬ tural relief, is provided for the identification of the sample holder and thus the sample located there¬ on. Said code can be made by laser, and preferably data related to the code (sample) are printed at the same time by means of a printer. The code is located on all sample holders in a predetermined angular po¬ sition in relation to the groove 23. Due to the fact that the code comprises a structural relief it can be read outside as well as inside the microscope, also when the sample holder has been coated with an electrically conducting thin layer. In accordance with modern technique the surface supporting the sample and also the annular circular surface sur- rounding said supporting surface, can be analyzed automatically by means of computer based picture analysis, which means that also the code can be read by such analysis. The code can comprise figures or letters or a combination thereof or a bar code or other code which cannot be optically read directly, i.e. it cannot be read and understood by ocular examination. When a code is provided which cannot be read optically it may be supplemented by a marking en clair which can be optically read and can be located on the cylindrical surface surrounding the annular circular surface.

All parts of the electrode except the filter disc should be made of an electrically conducting material. The base portion and the lock ring then preferably are made of an electrically conducting plastic material such as HD polyethylene, and this is true also as far as the revolving disc and the hub and pins thereof are concerned, but also a metal, e.g. aluminium, can be used for these parts. The code can be read in the scanning electron micro-

scope. Thus, it is easy to determine which sample holder is observed in the scanning electron micro¬ scope. The coordinates of interesting portions of the sample can be identified, and since the sample holder always is in the same rotational position on the revolving disc such portions can easily be found again, although the sample holder in question has been removed and has been put in again, because the rotational position of the sample holder on the pin is always the same.

Suction can be applied to all sample holders when the sample is being collected, viz. through the hub of the revolving disc by connecting said hub to a suction device. Through the connection to the hub an aeration can also take place when the electrode with the sample holders mounted thereon is exposed to a negative pressure at the metal coating and when used in the scanning electron microscope. This is important in case e.g. heavy protein layers have accumulated on the filter disc, which make the fil¬ ter disc impermeable to air. Without aeration bulging of the filter disc in that case may arise due to positive pressure in the cavity of the sample holder in connection with the metal coating or the analysis in the scanning electron microscope.

It is not necessary that the sample holders in the embodiment described are detachable. They may also be made integral with the revolving disc. In FIG. 5 there is shown another manner of effecting the aeration. The passage of each pin communicates with an aperture at the lower side of the revolving disc 26 instead of being connected to the hub. In this case it is necessary to deposit the sample on the filter disc by connecting each indi- vidual sample holder separated from the revolving

disc, to a suction device.

The analysis surface, i.e. the surface sup¬ porting the sample, of each sample holder is not necessarily air or liquid permeable; said surface can comprise a homogeneous surface. The collection of the sample on said surface then can be effected by a centrifugation method wherein particles, if any, entrained in the sample will sediment on the analysis surface. Alternatively, the analysis sur- face can be magnetic such that e.g. magnetic par¬ ticles can be collected on the surface by means of an electromagnetic field.

The use of the electrode/sample holder de¬ scribed is illustrated by the following example re- lating to the analysis of bacteria and virus, if any, in urine. In this case a holder having two filters of different pore size can be used since bacteria have a diameter of 1 μm and virus has a diameter ranging from 20 nm to 200 nm. The first filter then can comprise an analysis surface having a filter aperture size of 0.8 μ while the lower surface can comprise an analysis surface having a filter aperture size of 50 nm. Virus particles will pass through the upper analysis surface and will be recovered on the lower analysis surface. When the sample has been collected the analysis surfaces are detached and are mounted in the electrode, are coated with a gold/platinum layer or a coal layer in a so called sputter, and are examined in a scanning electron microscope.

Alternatively the particles searched for can be collected and enriched, respectively, by incubating micro spheres marked with antibodies, in the sample liquid, e.g. latex particles on the surface of which antibodies are located which are directed towards

the particle searched for. These micro spheres can be of a size ranging from 0.5 pri to 50 urn diameter. If the sample contains the virus searched for such virus will be bound to the micro spheres which then can be collected on the analysis surface which in this case can have a considerably larger pore size than in the previous example because it must prevent passage of micro spheres only and not of individual virus particles which have not been bound. If mag- netic micro spheres are used so as to bind to such spheres specific antibodies it is not necessary that the analysis surface is porous; the micro spheres will be collected on the analysis surface by means of an electromagnetic field.