JP2006518534A - Sample enclosure for a scanning electron microscope and its use - Google Patents

Abstract

An SEM sample container is a sample enclosure (100, 102) comprising an electron beam permeable and fluid impermeable membrane (132) and sealed to the membrane to form the sample enclosure together with the membrane. A sample enclosure comprising a surrounding enclosure, and a closure of the sample enclosure having a quick connect attachment (152) function for sealing engagement with the sample enclosure.

Description

  The present invention relates generally to SEM inspection of fluids containing samples, and more particularly to sample containers and inspection systems and methods of use thereof.

The following US patent documents are believed to represent the current state of the art.
US Patent No. 4,071,766 US Patent No. 4,720,633 US Patent No. 5,250,808 US Patent No. 5,326,971 US Patent No. 5,362,964 US Patent No. 5,412,211 US Patent No. 4,705,949 US Patent No. 5,945,672 US Patent No. 6,365,898 US Patent No. 6,130,434 US Patent No. 6,025,592 US Patent No. 5,103,103 US Patent No. 4,596,928 US Patent No. 4,880,976 US Patent No. 4,992,662 US Patent No. 4,720,622 US Patent No. 5,406,087 US Pat. No. 3,218,459 US Patent No. 3,378,684 US Patent No. 4,037,109 US Pat. No. 4,448,311 US Patent No. 4,115,689 US Pat. No. 4,587,666 US Patent No. 5,323,441 US Patent No. 5,811,803 US Pat. No. 6,452,177 US Patent No. 5,898,261 US Patent No. 4,618,938 US Patent No. 6,072,178 US Patent No. 6,114,695 US Patent No. 4,929,041

It is an object of the present invention to provide an apparatus, system and method for enabling SEM inspection of fluid containing a sample.
Thus, according to a preferred embodiment of the present invention, the SEM compatible sample container is a sample enclosure, which is an electron beam permeable and fluid impermeable membrane, sealed to the membrane and the sample together with the membrane. A sample enclosure comprising a surrounding enclosure forming an enclosure, and a closure of the sample enclosure having a quick connect attachment function for sealing engagement with the sample enclosure. Preferably, the function of the quick connection type attachment includes a bayonet type connection function. Furthermore, the surrounding enclosure is at least partially conductive. As an alternative or in addition, the SEM compatible sample container further comprises a pressure relief diaphragm associated with the sample enclosure.

  According to another preferred embodiment of the present invention, a SEM compatible sample container is provided further comprising at least one reference coordination indicator associated with the membrane. Preferably, the SEM compatible sample container further comprises at least one membrane support grid that supports the membrane and has a reference coordination indicator. In addition, the membrane is composed of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, “Perrodion”, “Colodion”, “Kapton”, “Form Bar”, “Vinyleck”, “Bat Bar”, “Pioroform”, It is formed from a material selected from the group consisting of “Parilene”, silicon dioxide, silicon monoxide, and carbon. As an alternative or in addition, the sample enclosure is pre-assembled and prepared to receive the liquid containing the sample, and following the preparation, the closure of the sample enclosure is easily sealed using the quick connect attachment feature. Can be linked.

  According to another preferred embodiment of the present invention, the SEM compatible sample container comprises a liquid sample enclosure, the liquid sample enclosure being sealed to the membrane with an electron beam permeable and fluid impermeable membrane. An ambient enclosure that forms the liquid sample enclosure with the membrane, the sample enclosure being capable of containing liquid at a depth that is not permeable by electrons having an energy level of less than 50 KeV.

  According to yet another preferred embodiment of the present invention, the SEM compatible sample container is a sample pan, an electron beam permeable fluid impermeable membrane, and sealed with the membrane together with the membrane. A sample enclosure comprising a surrounding enclosure forming a sample dish; and arranged around the sample dish to form an aperture for electrons to pass through the membrane with the interior of the dish And an outer enclosure.

  In accordance with yet another preferred embodiment of the present invention, an SEM compatible sample container includes an enclosure that forms an aperture for the passage of electrons and an electron beam transmissive and fluid impervious disposed within the enclosure. A sample pan with a membrane, the aperture being disposed relative to the membrane to communicate electrons with the interior of the enclosure through the membrane. Preferably, the sample pan is formed by a membrane together with the enclosure. Furthermore, the sample pan is formed by a membrane together with a separate pan wall arranged inside the enclosure. As an alternative or in addition, the separate dish wall is sealed to the membrane.

According to a further preferred embodiment of the present invention, the SEM compatible sample container is a sample pan assembly forming an aperture, the aperture communicating electrons through the aperture, the sample pan assembly being at least partially A sample non-rotating motion in engagement with the sample and the sample pan assembly comprising an electron beam transmissive and fluid impermeable membrane that forms a sample enclosure in general, thereby adjacent to the membrane A sample positioner configured to position the sample and a closure having a quick connect attachment function for sealing engagement with the enclosure;
The aperture is positioned relative to the membrane such that electrons are in communication therethrough with the sample adjacent to the membrane, the aperture penetrating the membrane. Preferably, the sample positioner comprises a flexible support element. In addition, the flexible support element comprises a cell support element.

  According to another preferred embodiment of the invention, the flexible support element comprises a cell growth element. Preferably, the flexible support element comprises a fluid filter element. In addition, the flexible support element comprises a membrane.

  According to yet another preferred embodiment of the invention, the flexible support element comprises an elastic membrane support. Preferably, the flexible support element is at least partially permeable to liquid. In addition, the sample positioner includes a spring.

  In accordance with a further preferred embodiment of the present invention, the SEM compatible sample container is a sample pan assembly that forms an aperture, the aperture communicating electrons through the aperture, the sample pan assembly being at least a portion of the sample pan assembly. The sample pan assembly comprising an electron beam transmissive and fluid impermeable membrane that forms a sample enclosure, and a pressure relief diaphragm associated with the sample pan assembly.

  According to a still further preferred embodiment of the present invention, the SEM compatible sample container is a sample pan assembly that forms an aperture, the aperture communicating electrons through the aperture, the sample pan assembly being at least The sample pan assembly comprising an electron beam transmissive and fluid impermeable membrane that partially forms a sample enclosure, and at least one reference configuration indicator associated with the membrane.

  In accordance with yet a further preferred embodiment of the present invention, a SEM-compatible pre-microscope multiple sample container system comprises a plurality of SEM compatible sample containers and a support for supporting a plurality of SEM compatible sample containers. A section. Preferably, the support part includes a light transmission part disposed below at least one of the membranes in the plurality of SEM compatible sample containers, whereby the plurality of SEM compatible sample containers are supported by the support part. In between, an optical microscope inspection can be performed on the sample in at least one of the SEM compatible sample containers. In addition, the SEM-compatible multi-sample container system prior to microscopic examination further includes a cover arranged to cover the support and a plurality of SEM-compatible sample containers supported by the support.

  According to another preferred embodiment of the invention, the support maintains the humidity of the sample within the plurality of SEM compatible sample containers while the plurality of SEM compatible sample containers are supported by the support. At least one liquid reservoir for holding a liquid that is useful for. Preferably, the SEM compatible multiple sample container is provided with a suction device and a pipette. In addition, the aspiration device is configured to prevent its physical engagement with the membranes of a plurality of SEM compatible sample containers upon its operative engagement with the support.

  According to yet another preferred embodiment of the invention, the pipette is provided with a collar element to prevent unintentional engagement between the pipette and the membrane. Preferably, the multiple sample container prior to microscopy is dimensioned to be compatible with conventional cell biology equipment. In addition, the support includes a holding function for removably holding individual containers of the plurality of SEM compatible sample containers relative to each other.

  According to yet another preferred embodiment of the present invention, the SEM system is a sample pan assembly that forms an aperture with the SEM, the aperture communicating electrons through the aperture, the sample pan assembly comprising: X-rays are received between the sample pan assembly comprising an electron beam transmissive and fluid impermeable membrane that at least partially forms a sample enclosure and a sample-containing liquid disposed in the sample enclosure during SEM inspection And an X-ray detector arranged as described above. Preferably, the SEM system further comprises a sample enclosure closure having a quick connect attachment function for sealing engagement with the sample enclosure. In addition, the quick connect attachment function includes a bayonet connection function.

  According to another preferred embodiment of the present invention, the sample enclosure is at least partially conductive. Preferably, the SEM system further comprises a pressure relief diaphragm associated with the sample enclosure. In addition, the SEM system further comprises at least one reference coordination indicator associated with the membrane.

  According to yet another preferred embodiment of the present invention, the SEM system further comprises at least one membrane support grid that supports the membrane and has a reference coordination indicator function. Preferably, the membrane is polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, “Perrodion”, “Colodion”, “Kapton”, “Form Bar”, “Vinyleck”, “Bat Bar”, “Pioform”, It is formed from a material selected from the group consisting of “Parilene”, silicon dioxide, silicon monoxide, and carbon. In addition, the sample enclosure is pre-assembled and prepared to receive the liquid containing the sample, and following the preparation, the sample enclosure closure is easily hermetically coupled using the quick connect attachment feature. be able to.

  In accordance with yet another preferred embodiment of the present invention, the SEM system is a SEM and a SEM compatible sample container having a sample enclosure, the sample enclosure comprising an electron beam transmissive and fluid impermeable membrane. A perimeter enclosure that forms a liquid sample enclosure with the membrane and has a quick connect attachment function for sealing engagement between the SEM compatible sample container and the sample enclosure A closure of the sample enclosure. Preferably, the function of the quick connection type attachment includes a bayonet type connection function. In addition, the surrounding enclosure is at least partially conductive.

  According to yet another preferred embodiment of the present invention, the SEM system further comprises a pressure relief diaphragm associated with the sample enclosure. Preferably. The SEM system further comprises at least one reference coordination indicator associated with the membrane. In addition, the SEM system further comprises at least one membrane support grid that supports the membrane and has a reference coordination indicator function.

  According to still another preferred embodiment of the present invention, the membrane comprises polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, “Perrodion”, “Colodion”, “Kapton”, “Form Bar”, “Vinyleck”. ”,“ Batbar ”,“ Pioloform ”,“ Parilene ”, silicon dioxide, silicon monoxide, and a material selected from the group consisting of carbon. Preferably, the sample enclosure is pre-assembled and prepared to receive the liquid containing the sample, and following the preparation, the sample enclosure closure is easily hermetically coupled using the quick connect attachment feature. Can do.

  According to yet another preferred embodiment of the present invention, a method for performing a scanning electron microscope examination is the step of placing a sample in a sample enclosure, the sample enclosure being electron beam transmissive. A sample enclosure having a fluid impermeable membrane, a surrounding enclosure that is sealed to the membrane to form the sample enclosure together with the membrane, and a quick connect attachment function for sealing engagement of the sample enclosure A step of sealing the sample enclosure with the sample enclosure closure, placing the sample enclosure in the electron beam, and analyzing the results of the interaction of the electron beam and the sample A process. Preferably, the method for performing a scanning electron microscope inspection further comprises removing liquid from the sample enclosure prior to the sealing step. In addition, the method for performing a scanning electron microscope inspection further comprises adding a liquid to the sample enclosure prior to the sealing step.

  According to yet another preferred embodiment of the present invention, a method for performing a scanning electron microscope examination comprises the step of culturing the sample in a sample enclosure. Preferably, the analysis of the result of the interaction between the sample and the electron beam comprises X-ray detection, detection of light in the ultraviolet to infrared range, detection of backscattered electrons, and detection of secondary electrons. Implemented by at least one of them.

  According to yet another preferred embodiment of the present invention, a method for performing a scanning electron microscope examination is the step of placing a sample in a sample enclosure, the sample enclosure being electron beam transmissive. A sample enclosure having a fluid impermeable membrane and a quick connect attachment function for sealing the engagement between the surrounding enclosure and the sample enclosure that is sealed to the membrane to form the sample enclosure. A closure, and placing the sample positioner arranged to position the sample adjacent to the membrane, sealing the sample enclosure with the closure of the sample enclosure, Placed inside, interaction between electron beam and sample Analyzing the results, including each step. Preferably, the method for performing a scanning electron microscope inspection further comprises removing liquid from the sample enclosure prior to the sealing step. In addition, the method for performing a scanning electron microscope inspection further comprises adding a liquid to the sample enclosure prior to the sealing step.

  According to another preferred embodiment of the present invention, the method for performing a scanning electron microscope examination further comprises culturing the sample in a sample enclosure. Preferably, the analysis of the result of the interaction between the sample and the electron beam includes X-ray detection, light detection in the ultraviolet to infrared range, backscattered electron detection, and secondary electron detection. Implemented by at least one. In addition, the sample positioner comprises a flexible support element.

  According to yet another preferred embodiment of the invention, the flexible support element comprises a cell support element. Preferably, the flexible support element comprises a cell growth element. In addition, the flexible support element comprises a fluid filter element.

  According to yet another preferred embodiment of the invention, the flexible support element comprises a membrane. Preferably, the flexible support element comprises an elastic membrane support. In addition, the flexible support element is at least partially permeable to liquid.

  According to yet another preferred embodiment of the invention, the quick connect attachment function comprises a bayonet connection function. Preferably, the sample enclosure is at least partially conductive. In addition, the sample positioner includes a spring.

  According to another preferred embodiment of the present invention, the method for performing a scanning electron microscope examination further comprises a pressure relief diaphragm associated with the sample pan assembly. Preferably, the method for performing a scanning electron microscope examination further comprises at least one reference coordination indicator associated with the membrane. In addition, the method for performing a scanning electron microscope examination further comprises at least one membrane support grid that supports the membrane and has a reference coordination indicator function.

  According to another preferred embodiment of the present invention, the membrane is composed of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, “Perrodion”, “Colodion”, “Kapton”, “Form Bar”, “Vinyleck”, It is formed from a material selected from the group consisting of “Bat Bar”, “Pioloform”, “Parilene”, silicon dioxide, silicon monoxide, and carbon. Preferably, the sample enclosure is pre-assembled and prepared to receive a liquid containing the sample, following which the closure can be easily hermetically coupled using a quick connect attachment feature.

According to still further preferred embodiments of the present invention, a method using an electron microscope provides a sample of an organic or polymeric material that is sensitive to damage induced by electron beam irradiation;
Each step includes adding a protective material to the sample to at least partially prevent damage induced by electron beam irradiation, and irradiating the sample and the protective material with an electron beam in an electron microscope. Preferably. Providing the sample comprises disposing the sample within a sample enclosure, the sample enclosure comprising an electron beam permeable and fluid impermeable membrane, sealed to the membrane and the sample together with the membrane. Form an enclosure.

  According to still further preferred embodiments of the present invention, a method of using an electron microscope is the step of placing a sample in a sample enclosure, the sample enclosure being sensitive to damage induced by electron beam irradiation. An electron beam permeable, fluid impermeable membrane and a surrounding enclosure sealed to the membrane to form the sample enclosure with the process, and damage induced by electron beam irradiation Each step includes adding a protective material that at least partially prevents to the sample and irradiating the sample and the protective material with an electron beam in an electron microscope. Preferably, the film is sensitive to electron beam irradiation induced damage resulting from electron beam irradiation on the film. In addition, the film is sensitive to electron beam irradiation induced damage due to electron beam irradiation of the sample.

  According to a still further preferred embodiment of the present invention, the open top microscope sample container is sealed to the light permeable fluid impermeable sample support having a thickness of less than 10 microns, And an open top surrounding enclosure that forms a top open microscope sample container with the sample support.

  The present invention will be understood and appreciated more fully from the following detailed description, taken in conjunction with the accompanying drawings, in which:

  Reference is now made to FIGS. 1A-5B, which are simplified exploded views, each directed to the opposite side, of an exploded SEM compatible sample container constructed and operative in accordance with a preferred embodiment of the present invention. As shown in FIGS. 1A and 1B, SEM-compatible sample containers are first and second indicated by reference numerals 100 and 102, arranged to improve the ease and speed of closure. Enclosure elements that are screwed together. Enclosure elements 100 and 102 improve ease and speed of closure by connecting protrusions 104 formed in first enclosure element 100 to bayonet-type sockets 106 formed in second enclosure element 102. It is configured as follows.

  The protrusion 104 preferably includes a first protrusion 108 and a second protrusion 110, and these protrusions include a first recess 112 and a second recess formed in the bayonet-type socket 106. 114. The initial engagement of the protrusion 108 with the recess 112 provides a loose engagement of the enclosure elements 100 and 102 to prevent, for example, accidental damage of the SEM compatible sample container during movement and handling. Subsequent engagement of the protrusion 108 with the recess 114 provides a tight engagement of the enclosure elements 100 and 102. Enclosure elements 100 and 102 are preferably cast from plastic and coated with a conductive metal coating.

  The first enclosure element 100 preferably forms a liquid sample enclosure and has a base surface 104 with a generally central aperture 106. The electron beam transmissive and fluid impermeable membrane subassembly 128, shown in detail in FIGS. 2A and 2B, is positioned against the aperture 106 as shown in FIGS. 3A, 3B and 5A, 5B. It covers and sits inside the enclosure element 100. A sample pan with a subassembly 108 suitably positioned within the enclosure element 100 is indicated by reference numeral 129, as shown in FIGS. 3A-5B.

  Still referring to FIGS. 2A and 2B, an electron beam transmissive and fluid impermeable membrane 130 is adhered to a grid support element 132 that forms a mechanical support grid 134 at its center by an adhesive. The membrane 130 preferably comprises a polyimide membrane, such as catalog number LWN00033, commercially available from Moxtec Corporation of the United States, UT, Orem. The grid 134 is not shown to scale in the drawing and is an asymmetric array to provide a reference configuration indicator function to assist the user in identifying the location of the sample region relative to the SEM compatible sample container. The stainless steel sheet commercially available from Slon, Kibbutzmergan Michael, Israel, is preferably formed by chemical etching. The adhesive is preferably catalog number 1193MVLV, commercially available from Diamix, 51 Greenwood Road, CT06790, USA.

  The liquid sample enclosure forming ring 136 is preferably attached to the electron beam permeable fluid impermeable membrane 130 by bonding, such as catalog number 1193 MVLV, commercially available from Diamix, 51 Greenwood Road, CT 06790, USA, for example. It is preferable that they are adhered. Ring 136 is preferably formed from PMMA (polymethylmethacrylate), such as catalog number 692106001000, commercially available from Ilpen, Barcelona, Spain, and has a height of about 2 mm in a volume of about 20 microliters. Preferably, a liquid sample enclosure is defined. Preferably, the ring 136 is configured to define a liquid sample enclosure 138 having an inclined wall, and the protrusion 144 of the first enclosure element 100 so that the ring 136 is seated steadily in the first enclosure element 100. A peripheral rim 140 is formed on the outer surface of the outer rim 140 that acts to engage a recess 142 formed in the outer surface.

  As an alternative, the membrane 130 may be made of polyamide, polyamide-imide, polyethylene, polypyrrole, “Perrodion”, “Colodion”, “Kapton”, “Form Bar”, “Vinyleck”, “Bat Bar”, “Pioroform”, “ It may be formed from "Parylene", silicon dioxide, silicon monoxide, or carbon, or any combination of those listed above, or any other suitable material.

  As shown in FIGS. 1A, 1B, 5A and 5B, the O-ring 146 is preferably disposed between the ring 136 and the inner surface 150 of the second enclosure element 102. The O-ring 146 acts to eliminate the need for the threaded engagement of the elements 100 and 102 to be a sealing engagement when the enclosure elements 100 and 102 are in close engagement.

  The second enclosure element 102 is preferably formed with a generally central stub 122 arranged to seat in a suitable recess (not shown) in the specimen stage of the scanning electron microscope. It is a special feature of the present invention that the container shown in FIGS. 1A to 10 operates in a size that fits the conventional stub recess of a conventional scanning electron microscope and does not need to be deformed whatever it is. is there. It will be appreciated that various forms and sizes of stubs can be provided to fit various scanning electron microscopes.

  Enclosure elements 100 and 102 extend radially to allow the container to be easily seated in a suitable multi-container holder and to assist the user to screw open and close the enclosure elements 100 and 102. Respective positioning and holding projections 154 and 156 are preferably provided. Preferably, the relative azimuthal arrangement of protrusions 154 and 156 on each enclosure element 100 and 102 is relative azimuthal direction between these protrusions, as shown in FIGS. 4A and 4B. Preferably, the alignment is indicative of the desired degree of screw closure between the elements.

It will be appreciated that in other embodiments of the invention, the sample pan may include enclosures 100 and 102.
Reference is now made to FIGS. 6A, 6B, and 6C, which are three cross-sectional views illustrating the operational aspects of the SEM compatible sample container of FIGS. FIG. 6A shows the container of FIGS. 1A-5B with a liquid sample 160 arranged in the configuration shown in FIG. 1B prior to closing enclosure elements 100 and 102. Note that the liquid sample does not flow out of the liquid sample enclosure 116 due to surface tension. An electron beam transmissive and fluid impermeable membrane 110 is shown in FIG. 6A to be substantially flat.

  FIG. 6B shows the container of FIG. 6A in an intermediate state following full engagement between enclosure elements 100 and 102 that create a seal of the liquid sample enclosure 138 from the surroundings. It can be seen that the electron beam transparent and fluid impermeable membrane 130 is outward due to the pressure build-up of the liquid sample enclosure 138 resulting from its sealing in this embodiment. The support grid 134 is shown to be substantially flat.

FIG. 6C shows the container of FIG. 6B when placed in a SEM degassing environment with a vacuum of typically 10 ⁻² to 10 ⁻⁶ mbar. In this environment, it can be seen that the electron beam transmissive and fluid impermeable membrane 130 and the support grid 134 are outwardly exposed to a greater degree than when placed in the ambient environment of FIG. 6B. Furthermore, it can be seen that the electron beam transmissive and fluid impermeable membrane 130 attempts to enter the gap through the gaps in the grating 134 to a greater degree than that produced in the ambient environment of FIG. 6B.

  Here, FIGS. 7A, 7B, 7C are simplified cross-sectional views of cell growth, liquid removal, liquid addition, sealing, and insertion into the SEM using the SEM compatible sample containers of FIGS. 1A-6C. See 7D and 7E. Referring to FIG. 7A, which illustrates a typical cell culture situation, the enclosure element 100 with the subassembly 128 disposed therein is in the arrangement shown in FIG. 1A, and the cells 162 in the liquid medium 164 are liquid samples. Located within the enclosure 138, these cells 162 lie in contact with an electron beam permeable, fluid impermeable membrane 130.

  FIG. 7B shows a state where liquid is removed from the liquid sample enclosure 138, typically by aspiration, and FIG. 7C shows a state where liquid is added to the liquid sample enclosure 138. Liquid removal and addition may be performed on the sample in the liquid sample enclosure 138 multiple times. Preferably, the device used for liquid removal and addition should be designed and equipped to prevent unintentional rupture of the electron beam permeable and fluid impermeable membrane 130.

  FIG. 7D shows the closed state of the container containing the cells 162 shown in FIG. 7C in the liquid medium 164. FIG. 7E shows the closed container inserted on the stage 166 of the SEM 168 in the configuration of FIG. 1B. It will be appreciated that there are SEMs whose container orientation is opposite to that shown in FIG. 7E.

  FIGS. 7A-7D illustrate a situation where at least a portion of the liquid containing the sample remains in contact with the electron beam transmissive and fluid impermeable membrane 130, regardless of the addition or removal of liquid with respect to the liquid sample enclosure 138. is doing. This situation may include a situation in which a portion of the sample is absorbed or otherwise adhered to the electron beam permeable, fluid impermeable membrane 130. Examples of liquids containing samples may include cells, cultures, blood, bacteria, and cell-free materials.

  FIG. 7 is a simplified cross-sectional view of a sample-containing liquid in contact with an electron beam permeable fluid impermeable membrane 130, sealed and inserted into an SEM using the SEM compatible sample container of FIGS. 1A-6C. Reference is made to FIGS. 8A, 8B and 8C. 8A-8C illustrate a situation where at least a portion of the liquid 170 containing the sample is in contact with the electron beam permeable fluid impermeable membrane 130 but is not adhered thereto. Examples of liquids containing samples include various emulsions and suspensions such as, for example, milk, cosmetic creams, paints, inks, and drugs in liquid form. It can be seen in FIGS. 8A-8B that the enclosure element 10 with the subassembly 128 disposed therein is in the configuration shown in FIG. 1A.

  FIG. 8B shows the closed state of the container containing the sample 170. FIG. 8C shows the closed container in the configuration of FIG. 1B inserted on the stage 166 of the SEM 168. It will be appreciated that there are SEMs whose container orientation is opposite to that shown in FIG. 8C.

  Reference is now made to FIG. 9, which is a simplified and cross-sectional view of SEM inspection of a sample using the SEM compatible sample container of FIGS. 1A-6C. As shown in FIG. 9, the container designated by reference numeral 171 has the electron beam 172 generated by the SEM passing through the electron beam permeable and fluid impermeable membrane 130 and the sample 174 in the container 171. It is arranged on the stage 166 of the SEM 168 so as to irradiate the liquid containing the liquid. Backscattered electrons from the sample 174 pass through an electron beam transparent and fluid impermeable membrane 130 and are detected by a detector 176 that forms part of the SEM 168. For example, one or more additional detectors such as secondary electron detector 178 may be provided. An X-ray detector (not shown) may be provided for detecting X-ray radiation emitted by the sample 174 due to electron beam excitation. A cathodoluminescent detector (not shown) may be provided for detecting radiation emitted by the sample 174 due to electron beam excitation.

  Reference is now additionally made to FIG. 10, which schematically shows details of electron beam interaction with a sample 174 in a container 171 according to a preferred embodiment of the present invention. As shown in FIG. 10, the present invention enables high-contrast image formation of feature groups distinguished from each other by their average atomic numbers. In FIG. 10, the cell nucleus 180, having a relatively high average atomic number, is shown to backscatter electrons more than the surrounding nucleus 182.

  It should be noted that according to a preferred embodiment of the present invention, imaging within a sample up to about 2 microns deep can be achieved for electrons having an energy level lower than 50 KeV, as shown in FIG. The cell nuclei 180 located below the electron beam permeable and fluid impermeable membrane 130 are imaged.

  Here, a simplified exploded view, each directed to the opposite side, of a disassembled scanning electron microscope (SEM) compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention, FIG. Refer to FIGS. 11A to 15B. As shown in FIGS. 11A and 11B, the SEM compatible sample container connects the protrusion 204 formed in the first enclosure element 200 to the bayonet-type socket 206 formed in the second enclosure element 202. To the first and second interengaging enclosure elements, indicated by reference numerals 200 and 202, arranged to improve the ease and speed of closure.

  The protrusion 204 preferably includes a first protrusion 208 and a second protrusion 210, which are a first recess 212 and a second recess formed in the bayonet-type socket 206. Communicate with 214. The initial engagement of the protrusion 208 with the recess 212 provides a loose engagement of the enclosure elements 200 and 202 to prevent, for example, accidental damage of the SEM compatible sample container during movement and handling. Subsequent engagement of the protrusion 208 with the recess 214 provides a tight engagement of the enclosure elements 200 and 202. Enclosure elements 200 and 202 are preferably cast from plastic and coated with a conductive metal coating.

  The first enclosure element 200 preferably forms a liquid sample enclosure and has a base surface 224 having a generally central aperture 226. The electron beam transmissive and fluid impermeable membrane subassembly 228, shown in detail in FIGS. 12A and 12B, is positioned against the aperture 226 as shown in FIGS. 13A, 13B and 15A, 15B. It covers and sits inside the enclosure element 200. A sample pan with a subassembly 228 appropriately positioned within the enclosure element 200 is indicated by reference numeral 229, as shown in FIGS. 13A-15B.

  Still referring to FIGS. 12A and 12B, an electron beam permeable, fluid impermeable membrane 230 is adhered to the mechanical support grid 232 by an adhesive. The membrane 230 preferably comprises a polyimide membrane, such as catalog number LWN00033, commercially available from Moxtec Corporation of the United States, UT, Orem. The support grid 232 forms a mechanical support grid 234 at its center. The grid 234 is not shown to scale in the drawing and the asymmetric array of apertures to provide a reference configuration identification function to assist the user in identifying the location of the sample region relative to the SME compatible sample container. Preferably, it is formed by photochemical etching of a stainless steel sheet commercially available from Slon, Kibbutz Margan Michael, Israel.

  The liquid sample enclosure forming ring 236 is preferably electron beam transmissive and fluid impermeable with an adhesive such as catalog number 1193 MVLV, commercially available from the United States, CT06790, Torrington, 51 Greenwood Road, Diamix. Bonded to the membrane 240. Ring 236 is preferably formed from PMMA (polymethylmethacrylate), such as catalog number 692106001000, commercially available from Ilpen, Barcelona, Spain, and has a height of about 2 mm in a volume of about 20 microliters. Preferably, a liquid sample enclosure is defined. Preferably, the ring 236 is configured to define a liquid sample enclosure 238 having an inclined wall. Furthermore, the ring 246 acts on its outer surface to engage a recess 240 formed in the protrusion 241 of the first enclosure element 200 to ensure that the ring 236 is seated on the first enclosure element 200. A peripheral rim 239 is preferably formed.

  As shown in FIGS. 11A, 11B, 15A and 15B, the diaphragm 242 is preferably disposed between the ring 236 and the inner surface 244 of the second enclosure element 202. Preferably, the diaphragm 242 is integrally formed from an O-ring portion 246, and an expandable sheet portion 248 is sealed in the O-ring portion 246. Diaphragm 242 is preferably molded from silicone rubber having a Shore hardness of about 50, and sheet portion 248 preferably has a thickness of 0.2 to 0.3 mm. Diaphragm 242 eliminates the need for the engagement of elements 200 and 202 to be a sealing engagement when enclosure elements 200 and 202 are in close engagement, and its dynamic and static pressures. Operates to provide relief.

  The second enclosure element 202 is preferably formed with a generally central stub 252 having a through-bore 253 arranged to seat in a suitable recess (not shown) in the scanning electron microscope specimen stage. . The bore 253 allows the diaphragm 242 to provide pressure relief by forming a fluid communication channel between one side of the diaphragm 242 and the environment in which the (SEM) compatible sample container is located. . It is a special feature of the present invention that the container shown in FIGS. 11A to 20 operates in a size that fits a conventional stub recess of a conventional scanning electron microscope and does not need to be deformed whatever it is. is there. It will be appreciated that various forms and sizes of stubs can be provided to fit various scanning electron microscopes.

  Enclosure elements 200 and 202 each extend radially to allow the container to be easily seated in a suitable multi-container holder and to assist the user in opening and closing enclosure elements 200 and 202, respectively. The positioning and holding projections 254 and 256 are preferably provided. Preferably, the relative azimuthal placement of protrusions 254 and 256 on each enclosure element 200 and 202 is relative azimuthal direction between these protrusions, as shown in FIGS. 14A and 14B. Preferably, the alignment is indicative of intimate engagement between the elements.

  Reference is now made to FIGS. 16A, 16B, and 16C, which are three cross-sectional views illustrating the operational aspects of the SEM compatible sample container of FIGS. 11A-15B, respectively, in three stages of operation. FIG. 16A shows the container of FIGS. 11A-15B storing liquid sample 260 arranged in the configuration shown in FIG. 11B prior to closing enclosure elements 200 and 202. Note that the liquid sample does not flow out of the liquid sample enclosure 238 due to surface tension. An electron beam transparent and fluid impermeable membrane 230 is shown in FIG. 16A to be substantially flat.

  FIG. 16B shows the container of FIG. 16A in an intermediate state following full engagement between enclosure elements 200 and 202 that create a seal of the liquid sample enclosure 238 from the surroundings. It can be seen that the diaphragm 242 is outward due to the pressure buildup of the liquid sample enclosure 238 resulting from its sealing in this embodiment. In this embodiment, the electron beam permeable fluid impermeable membrane 230 and its support grid 212 are also turned outward due to pressure buildup in the liquid sample enclosure 238 resulting from its sealing in this embodiment. However, due to the action of the diaphragm 242, it is only to a much smaller extent. This can be understood by comparing FIG. 16B and FIG. 6B. Support grid 234 is shown to be substantially flat.

FIG. 16C shows the container of FIG. 16B when placed in a SEM degassing environment with a vacuum of typically 10 ⁻² to 10 ⁻⁶ mbar. In this environment, the diaphragm 242 is outwardly exposed to a greater degree than in the ambient environment of FIG. 16B, and the electron beam permeable, fluid impermeable membrane 230 and the support grid 234 provide the ambient environment of FIG. 16B. It can be seen that it is outward to a greater degree than when it is placed in, but due to the action of the diaphragm 242, it is only slightly less than in the embodiment of FIG. 6C. . This can be understood by comparing FIG. 16C with FIG. 6C.

  It should be noted that the electron beam permeable and fluid impermeable membrane 230 attempts to enter the gap through the gaps in the grid 234 to a greater degree than that produced in the surrounding environment of FIG. 16B, but the action of the diaphragm 242 It can be seen that due to this, it is only to a much lesser extent than in the embodiment of FIG. 6C. This can be understood by comparing FIG. 16C with FIG. 6C.

  Here, FIGS. 17A, 17B, and 17C are simplified cross-sectional views of cell growth, liquid removal, liquid addition, sealing, and insertion into the SEM using the SEM compatible sample containers of FIGS. Reference is made to 17D and 17E. Referring to FIG. 17A, which is identical to FIG. 7A and shows a typical cell culture situation, enclosure element 200 with subassembly 228 disposed therein is in the configuration shown in FIG. The cells 262 are disposed within the liquid sample enclosure 238 and the cells 262 are shown lying in contact with the electron beam permeable, fluid impermeable membrane 230.

  FIG. 17B, identical to FIG. 7B, shows the removal of liquid from the liquid sample enclosure 238, typically by aspiration, and FIG. 17C, identical to FIG. 7C, shows the addition of liquid to the liquid sample enclosure 238. . It will be appreciated that multiple removals and additions of liquid can occur for a sample in the liquid sample enclosure 238. Preferably, the device used for liquid removal and addition should be designed and equipped to prevent unintentional rupture of the electron beam transmissive and fluid impermeable membrane 230.

  FIG. 17D shows the closed state of the container containing the cells 262 shown in FIG. 17C in the liquid medium 264. FIG. 17E shows the closed container inserted on stage 266 of SEM 268 in the configuration of FIG. 11B. It will be appreciated that there are SEMs whose container orientation is opposite to that shown in FIG. 17E.

  17A-17D illustrate a situation in which at least a portion of the liquid containing the sample remains in contact with the electron beam transmissive and fluid impermeable membrane 230, regardless of the addition or removal of liquid with respect to the liquid sample enclosure 238. is doing. This situation may include a situation in which a portion of the sample is absorbed or otherwise adhered to the electron beam transmissive and fluid impermeable membrane 230. Examples of liquids containing samples may include cells, cultures, blood, bacteria, and cell-free materials.

  FIG. 17 is a simplified cross-sectional view of sample-containing liquid in contact with an electron beam permeable fluid impermeable membrane 230, sealed and inserted into the SEM using the SEM compatible sample container of FIGS. 11A-16C. Reference is made to FIGS. 18A, 18B and 18C. FIGS. 18A-18C illustrate a situation where at least a portion of the liquid 270 containing the sample is in contact with the electron beam permeable fluid impermeable membrane 230 but is not adhered thereto. Examples of liquids containing samples include various emulsions and suspensions such as, for example, milk, cosmetic creams, paints, inks, and drugs in liquid form. 18A-18B, it can be seen that the enclosure element 200 with the subassembly 208 disposed therein is in the configuration shown in FIG. 11A. FIG. 18A is the same as FIG. 8A.

  FIG. 18B shows the closed state of the container containing the sample 270. FIG. 18C shows the closed container in the configuration of FIG. 11B inserted on stage 266 of SEM 268. It will be appreciated that there are SEMs whose container orientation is opposite to that shown in FIG. 18C.

  Reference is now made to FIG. 19, which is a simplified and cross-sectional view of a sample SEM inspection using the SEM compatible sample container of FIGS. 11A-16C. As shown in FIG. 19, the container designated by reference numeral 271 is such that the electron beam 272 generated by the SEM passes through the electron beam transmissive and fluid impermeable membrane 230 so that the sample 274 in the container 271 is present. Is placed on the stage 266 of the SEM 268 so as to irradiate the liquid containing the liquid. Backscattered electrons from the sample 274 pass through an electron beam transparent and fluid impermeable membrane 230 and are detected by a detector 276 that forms part of the SEM 268. One or more additional detectors may be provided, such as a secondary electron detector. An X-ray detector (not shown) may be provided for detecting X-ray radiation emitted by the sample 274 due to electron beam excitation. A cathodoluminescent detector (not shown) for detecting radiation emitted by the sample 274 due to electron beam excitation may also be provided.

  Reference is now additionally made to FIG. 20, which schematically shows details of electron beam interaction with a sample 274 in a container 270 according to a preferred embodiment of the present invention. In addition, as shown in FIG. 20, the present invention enables high-contrast image formation of feature groups distinguished from each other by their average atomic numbers. In FIG. 20, the cell nucleus 280, having a relatively high average atomic number, is shown to backscatter electrons more than the surrounding nucleus 282.

  Note that, according to a preferred embodiment of the present invention, imaging inside samples up to about 2 microns deep can be achieved for electrons having energy levels below 50 KeV, as shown in FIG. The cell nuclei 280 located below the membrane 230 that is transparent to the electron beam and impermeable to fluid are imaged.

  Reference is now made to FIGS. 21A and 21B, which are simplified exploded views of a multiple sample holder prior to microscopy for use with an SEM compatible sample container of the type shown in FIGS. 1A-20. Reference is further made to FIGS. 21A and 21B, which are simplified diagrams showing the multiple sample holders before microscopy, in an uncovered state and in a covered and assembled state, respectively. As shown in FIGS. 21A and 21B, the multiple sample holder before microscopic examination preferably includes a base 300, a top element 302, and a cover 304. Preferably, a cover 304 is provided to maintain sterility within the multiple sample holder prior to microscopy.

  Base portion 300 is preferably injection molded from a plastic material to form a row of container support receptacles 306. Each container support housing portion 306 is preferably formed by a recess 308 having a light transparent bottom wall. An optical microscope inspection can be performed through the light transparent bottom wall. An enclosure element indicated by reference numeral 100 in FIGS. 1A-8C and indicated by 200 in FIGS. 11A-15B, adjacent to each recess 308, indicated by reference numeral 154 in FIGS. 1A-5B. Preferably, a pair of mutually aligned pairs of upstanding inter-spaced protrusions 310 are formed that are arranged to receive the projected protrusions, thereby fixing the azimuthal alignment.

  The base 300 preferably forms a plurality of liquid reservoirs 312 that are adapted to hold liquid used to maintain a desired level of humidity within the multiple sample holder prior to microscopy. The base part 300 is preferably formed with a floor part 320.

  A cover 304 is provided to maintain sterility within the multiple sample holder prior to microscopy. The cover 304 is preferably transparent to light. The pre-microscope multiple sample holder of FIGS. 21A-22B is preferably dimensioned to be compatible with conventional cell biology equipment such as optical microscopes, centrifuges and automatic positioning devices. A preferred dimension is 85 mm × 127 mm.

  Reference is now made to FIGS. 22A, 22B and 22C, which are simplified illustrations of the multiple sample holder of FIGS. 21A-22B prior to microscopic examination, with a suction device and pipette. Referring to FIG. 22A, it is shown that the suction device indicated by reference numeral 350 includes a manifold 352 coupled to a suction source via a conduit 354. Manifold 352 is preferably in communication with a linear array of uniformly spaced needles 356. Manifold 352 has an orifice 357 formed at one end thereof to provide improved and independent suction action of individual needles 356.

  A pair of spacers 358 are attached to the manifold 352 or are integrally formed with the manifold. The spacer 358 is arranged in line with the linear row of needles 356. These spacers 358 ensure that the needle 356 does not engage the electron beam permeable fluid impermeable membrane indicated by reference numeral 130 in FIGS. 1A-10 and reference numeral 230 in FIGS. 11A-20. Make sure. Spacer 358 is on the side of the sample pan designated by reference numeral 129 in FIGS. 3A-5B and by reference numeral 229 in FIGS. 13A-15B to prevent needle 356 from expelling the sample in the sample pan. Engages with recess 359 to be aligned along the portion.

  As shown in FIG. 22A, the container support positions 306 are arranged in a linear row on the multiple sample holder before microscopy. Thus, as shown in FIG. 22B, for each row, four of the needles 356 engage the sample pan.

  FIG. 22C shows a state in which liquid is added to each sample pan using a conventional pipette 360. In order to prevent unintentional engagement of the pipette with the electron beam permeable, fluid impermeable membrane, indicated by reference numeral 130 in FIGS. 1A-10 and 230 in FIGS. 11A-20. , May be provided for use in conjunction with pipette 360.

  Reference is now made to FIG. 23, which is a simplified illustration of an SEM-based sample inspection system constructed and operative in accordance with a preferred embodiment of the present invention. As shown in FIG. 23, a plurality of pre-microscopic multiple sample holders 600 each include a number of SEM compatible sample containers 602 of the type shown in FIGS. 1A-20, within the bacterial incubator 604. Shown in the deployed state. Preferably, to identify the sample of interest, light microscopy of the sample in the container 602 is performed with the container 602 mounted in the holder 600 as indicated by reference numeral 606. Preferably, an inverted optical microscope 608 is used for this purpose.

  Preferably, an automatic positioning system, such as a robot arm as shown, is used to transport the pre-microscopic multiple sample holder 600 and container 602 through the system. It will be appreciated that manual intervention may be used as appropriate in one or more stages.

  Thereafter, the individual containers 602 are removed from the holder 600 and placed on the removable electron microscope specimen stage 610 and then introduced into the scanning electron microscope 612. The resulting image is visually inspected by an operator and / or analyzed by conventional image analysis functions typically implemented in computer 614.

  FIGS. 24A-28B are simplified exploded views, each directed to the opposite side, of an exploded scanning electron microscope (SEM) compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention. Refer to As shown in FIGS. 24A and 24B, the SEM-compatible sample container is provided with first and second enclosure elements indicated by reference numbers 1100 and 1102, respectively, to improve the ease and speed of closure. And connecting elements 1103 arranged. Enclosure elements 1100 and 1102 and connecting element 1103 are preferably molded from plastic and coated with a conductive metal coating.

  The first enclosure element 1100 preferably forms a sample enclosure and has a base surface 1104 with a generally central aperture 1106. The electron beam transmissive and fluid impermeable membrane subassembly 1108, shown in detail in FIGS. 25A and 25B, is positioned opposite the aperture 1106 as shown in FIGS. 26A, 26B and 28A, 28B. It covers and sits inside the enclosure element 1100. A sample pan with a subassembly 1108 suitably positioned within the enclosure element 1100 is indicated by reference numeral 1109, as shown in FIGS. 26A-28B.

  Still referring to FIGS. 25A and 25B, an electron beam transmissive and fluid impermeable membrane 1110 is adhered to the grid support element 1111 with an adhesive. The membrane 1110 preferably comprises a polyimide membrane, such as catalog number LWN00033, commercially available from Moxtec Corporation of the United States, UT, Orem. The grid support element 1110 forms a machine support grid 1112 at its center. The grid 1112 is not shown to scale in the drawing and is an asymmetrical aperture to provide a reference coordination indicator function to help the user identify the location of the sample region relative to the SEM compatible sample container. Preferably, it is configured to form an array, preferably formed by photochemical etching of a stainless steel sheet commercially available from Chelon of Kibbutzmergan Michael, Israel.

  The sample enclosure forming ring 1114 is preferably adhered to the electron beam transmissive and fluid impermeable membrane 1110 by an adhesive. Examples of the adhesive are commercially available from Diamix, Inc., USA, CT06790, Torrington, 51 Greenwoods Road, for example, catalog number 1193MVLV. Ring 1114 is preferably formed from PMMA (polymethyl methacrylate), such as catalog number 692106001000, which is commercially available from Ilpen, Barcelona, Spain, and has a height of about 2 mm in a volume of about 20 microliters. Preferably, a liquid sample enclosure is defined. Preferably, the ring 1114 is operative to engage a recess 1119 formed in the protrusion 1120 of the first enclosure element 1100 to ensure seating of the ring 1114 within the first enclosure element 1100. A rim 1118 is formed on the outer surface thereof.

  As shown in FIGS. 24A, 24B, 28A, and 28B, the first O-ring 1121 is preferably disposed between the inner surface 1122 of the second enclosure element 1102 and the connecting element 1103. The second O-ring 1126 is preferably disposed between the connecting element 1103 and the ring 1114 of the subassembly 1108. To eliminate the need for sealing engagement of enclosure elements 1100 and 1102 and connection element 1103, O-rings 1121 and 1126 act when enclosure elements 1100 and 1102 and connection element 1103 are in a tight engagement.

  The connecting element 1103 preferably has a recess 1128. The connection element 1103 is formed with a protrusion 1130 shown in FIGS. 28A and 28B, and the protrusion protrudes into the recess 1128.

  The positioner 1132 is preferably composed of two upstanding flexible protrusions 1134, each comprising a ridge 1136 formed at the end 1138 of the protrusion 1134. The positioner 1132 is preferably molded from plastic. As shown in FIGS. 28A and 28B, the protrusions 1134 press against each other when inserted into the recess 1128 of the connection element 1103 and once the ridge 1136 is seated on the protrusion 1130 of the connection element 1103, Snap back to upright position.

  The positioner 1132 is preferably provided with positioning and holding projections 1140 extending from the rim 1142 and extending in the respective radial directions. The positioning and holding projection 1140 is seated in the groove 1117 of the ring 1114 to prevent the positioner 1132 from rotating in the ring 1114.

  The coil spring 1144 is disposed on the positioner 1132 between the rim 1142 and the ridge 1136 of the protrusion 1134. The spring 1144 is preferably formed from hardened stainless steel.

  The positioner 1132 and the spring 1144 contact the electron beam transparent and fluid impermeable membrane 1110 to bring the non-liquid sample upward when the enclosure elements 1100 and 1102 and the connecting element 1103 are in intimate engagement. Acts to move.

  The first enclosure element 1100 engages the connection element 1103 by connecting a protrusion 1154 formed in the first enclosure element 1100 to a bayonet-type socket 1156 formed in the connection element 1103.

  The projecting portion 1154 includes a first projecting portion 1158 and a second projecting portion 1160, and these projecting portions are formed in the bayonet-type socket 115, a first recess 1162 and a second recess. 1164. The initial engagement of the protrusion 1158 with the recess 1162 is loose, for example, between the first enclosure element 1100 and the connection element 1103 to prevent accidental damage to the SEM compatible sample container during movement and handling. Provide engagement. Subsequent engagement of protrusion 1158 with recess 1164 provides a tight engagement of enclosure element 1100 and connecting element 1103.

  The second enclosure element 1102 engages the connection element 1103 by connecting a protrusion 1154 formed on the connection element 1103 to a bayonet-type socket 1176 formed on the second enclosure element 1102.

  The projecting portion 1174 includes a first projecting portion 1178 and a second projecting portion 1180, and these projecting portions are formed in a bayonet-type socket 1176, a first recess 1182, and a second recess 1184. The initial engagement of the protrusion 1178 with the recess 1182 loosens the second enclosure element 1102 and the connecting element 1103 to prevent, for example, accidental damage of the SEM compatible sample container during movement and handling. Provide engagement. Subsequent engagement of the protrusion 1178 with the recess 1184 provides a tight engagement between the enclosure element 1102 and the connection element 1103.

  The second enclosure element 1102 is preferably formed with a generally central stub 1190 arranged to seat in a suitable recess (not shown) in the specimen stage of the scanning electron microscope. It is a special feature of the present invention that the container shown in FIGS. 24A-33 works in a size that fits a conventional stub recess in a conventional scanning electron microscope and does not need to be deformed whatever it is. is there. It will be appreciated that various forms and sizes of stubs can be provided to fit various scanning electron microscopes.

  Enclosure elements 1100 and 1102 and connecting element 1103 allow the container to be easily seated in a suitable multi-container holder and further allows the user to open and close enclosure elements 1100 and 1102 and connecting element 1103. To assist, it is preferable to provide respective positioning and holding projections 1194, 1196 and 1198 extending radially. Preferably, the relative azimuthal arrangement of the respective protrusions 1194, 1196 and 1198 on each enclosure element 1100 and 1102 and connecting element 1103 is such that the protrusions are shown in FIGS. 27A and 27B. It is preferred that the relative azimuthal alignment between them indicates a tight engagement between the elements.

  Reference is now made to FIGS. 29A, 29B, and 29C, which are three cross-sectional views illustrating the operational aspects of the SEM compatible sample container of FIGS. 24A-28B, respectively, in three stages of operation. FIG. 29A shows the container of FIGS. 24A-28B storing tissue samples 1260 arranged in the configuration shown in FIG. 24B prior to closing enclosure elements 1100 and 1102 and connecting element 1103. FIG. An electron beam transmissive and fluid impermeable membrane 1110 is shown in FIG. 19A to be substantially flat.

  FIG. 29B shows the container of FIG. 29A in an intermediate state following full engagement between enclosure elements 1100 and 1102 and connecting element 1103 that creates a seal of tissue sample enclosure 1116 from the surroundings. Note that the tissue sample 1260 is in intimate contact with the electron beam transmissive and fluid impermeable membrane 1110 due to the force exerted by the positioner 1132. It can be seen that the electron beam permeable fluid impermeable membrane 1110 is outward due to the pressure buildup of the tissue sample enclosure 1116 resulting from its sealing in this embodiment. Support grid 1112 is shown to be substantially flat.

FIG. 29C shows the container of FIG. 29B when placed in a SEM degassing environment with a vacuum of typically 10 ⁻² to 10 ⁻⁶ mbar. In this environment, it can be seen that the electron beam permeable and fluid impermeable membrane 1110 and the support grid 1112 are more outwardly exposed than when placed in the surrounding environment of FIG. 29B. Furthermore, it can be seen that the electron beam transmissive and fluid impermeable membrane 1110 attempts to enter the gap through the gaps in the grating 1112 to a greater degree than that produced in the surrounding environment of FIG. 36B.

  Reference is now made to FIG. 30, which is a simplified and cross-sectional view showing the tissue containing the sample and insertion into the SEM using the SEM compatible sample container of FIGS. 24A-29C. FIG. 30 shows the closed container in the configuration of FIG. 24B inserted into the stage 1264 of the SEM 1266. It will be appreciated that there are SEMs whose container orientation is the opposite of that shown in FIG.

  Here, various operational configurations of the SEM compatible sample containers of FIGS. 24A-29B are shown in various stages of operation and insertion into the SEM using the SEM compatible sample containers, FIGS. 31A, 31B, Reference is made to 31C, 31D and 31E.

  FIG. 31A illustrates a plurality of flexibility operating to support particles or cells, such as a cell support element, cell growth element, fluid filter element, and cell stage 1268, typically placed in an incubator 1270. The sex support element is shown. The cell stage 1268 serves to allow the growth of cells on the stage or, alternatively, to allow adherence of the growing cells on the stage. Each cell stage 1268 is preferably formed by a porous membrane 1272 surrounded by a ring 1274 and supports a sample 1276 containing cells 1278. The porous membrane 1272 is preferably formed from polyester, such as catalog number 3470, Townswell plate, commercially available from Corning, Acton, USA. Cells 1278 are preferably supported by a porous membrane 1272 on the basal side of cells 1278 in a liquid medium. Porous membrane 1272 serves to maintain the desired level of humidity in the sample during the phase of operation, such as during incubation in incubator 1270, as shown in FIG. 31A.

  It will be appreciated that the cells 1278 or particles can be deposited on the flexible support element by adsorbing or filtering the fluid containing the cells or particles through the porous membrane 1272.

  As shown in FIG. 31A, a molecule 1279, such as a molecule of a cell 1278 or an externally added molecule, is preferably provided on the top side of the cell 1278. Preferably, the molecule 1279 is linked to a high electron density structure such as a gold colloid that can be visualized with an electron microscope.

  FIG. 31B supports cells 1278, as specified below, except that it includes a cell stage 1268 mounted on a platform 1282 preferably formed from a non-rigid material. An SEM compatible sample container 1280 that is identical to the container is shown. Container 1280 is arranged in the configuration shown in FIG. 24B prior to closure of enclosure elements 1100 and 1102 and connecting element 1103. The cell growth platform 1282 is mounted on a suitably configured positioner 1284 corresponding to the positioner 1132 of the example of FIGS. 24A-30. Typically, the cells 1278 grow on the cell stage 1268 in the incubator 1270 even when the cell stage 1268 is not mounted on the platform 1282. The mounting of the cell stage 1268 on the platform 1282 and the mounting of the plot form 1282 with the cell stage 1268 on the positioner 1284 are typically performed immediately before the SEM examination is performed.

  FIG. 31C shows container 1280 of FIG. 31B immediately following full engagement between enclosure elements 1100 and 1102 and connecting element 1103 that creates a seal of the cell sample enclosure designated by reference numeral 1286 from the surroundings. Is shown. Note that the sample containing the cells 1278 is in intimate contact with the electron beam permeable and fluid impermeable membrane 1110 due to the force exerted by the positioner 1284. It can be seen that the electron beam permeable fluid impermeable membrane 1110 is outward due to the pressure buildup of the tissue sample enclosure 1286 resulting from its sealing in this embodiment. Support grid 1112 is shown to be substantially flat.

FIG. 31D shows the container of FIG. 31C when placed in a SEM degassing environment with a vacuum of typically 10 ⁻² to 10 ⁻⁶ mbar. In this environment, it can be seen that the electron beam permeable and fluid impermeable membrane 1110 and the cell stage 1268 are more outwardly exposed than when placed in the surrounding environment of FIG. 31C. Furthermore, it can be seen that the electron beam transmissive and fluid impermeable membrane 1110 attempts to enter the gap through the gap in the grating 1112 to a greater degree than that produced in the surrounding environment of FIG. 31C. It can be seen that in this environment the grid 1112 is also slightly outward.

  FIG. 31E shows the closed container 1280 in the configuration of FIG. 24B inserted into the stage 1264 of the SEM 1266. It will be appreciated that there are SEMs whose container orientation is opposite to that shown in FIG. 31E.

  Reference is now made to FIG. 32 which is a simplified and cross-sectional view of a sample SEM inspection using the SEM compatible sample container of FIGS. 24A-30. As shown in FIG. 32, the container designated by reference numeral 1290 passes through a membrane 1110 (FIGS. 24A-31D) in which an electron beam 1292 generated by an SEM passes through an electron beam transparent and fluid impermeable membrane. , Placed on the stage 1264 of the SEM 1266 so that the tissue including the sample 1294 in the container 1290 is irradiated. Backscattered electrons from the sample 1294 pass through an electron beam transparent and fluid impermeable membrane 1110 and are detected by a detector 1296 that forms part of the SEM. One or more additional detectors may be provided such as, for example, secondary electron detector 1298. An x-ray detector (not shown) may be provided for detecting x-ray radiation emitted by the sample 1294 due to electron beam excitation. A cathodoluminescent detector (not shown) may be provided for detecting radiation emitted by the sample 1294 due to electron beam excitation.

  Reference is now additionally made to FIG. 33, which schematically shows details of electron beam interaction with a sample 1294 in a container 1290 according to a preferred embodiment of the present invention. Note that the present invention enables high-contrast image formation of feature groups distinguished from each other by their average atomic number, as shown in FIG. In FIG. 33, a cell nucleus 1300 having a relatively high average atomic number is shown to backscatter electrons more than the surrounding nucleoplasm 1302.

  It should be noted that according to a preferred embodiment of the present invention, imaging inside samples up to about 2 microns deep can be achieved for electrons having energy levels below 50 KeV, as shown in FIG. The cell nucleus 1300 located below the membrane 1110 that is electron beam permeable and fluid impermeable is imaged.

  Here, a simplified exploded view, each directed to the opposite side, of a disassembled scanning electron microscope (SEM) compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention, FIG. Reference is made to FIGS. 34A to 38B. As shown in FIGS. 34A and 34B, the SEM compatible sample container improves the ease and speed of closure with the first and second enclosure elements indicated by reference numerals 2100 and 2102 respectively. And connecting elements 2103 arranged as described above. Enclosure elements 2100 and 2102 and connecting element 2103 are preferably molded from plastic and coated with a conductive metal coating.

  The first enclosure element 2100 preferably has a base surface 2104 that forms a sample enclosure and has a generally central aperture 2106. The electron beam transmissive and fluid impermeable membrane subassembly 2108, shown in detail in FIGS. 35A and 35B, is positioned opposite the aperture 2106 as shown in FIGS. 36A, 36B and 38A, 38B. It covers and sits inside the enclosure element 2100. A sample pan with a subassembly 2108 suitably positioned within the enclosure element 2100 is indicated by reference numeral 2109, as shown in FIGS. 36A-38B.

    Still referring to FIGS. 35A and 35B, an electron beam transmissive and fluid impermeable membrane 2110 is adhered to the grid support element 2111 with an adhesive. The membrane 2110 is preferably a polyimide membrane, such as catalog number LWN00033, commercially available from Moxtec Corporation of the United States, UT, Orem. The grid support element 2111 forms a mechanical support grid 2112 at the center thereof. The grid 2112 is not shown to scale in the drawing and is an asymmetric array to provide a reference configuration indicator function to assist the user in identifying the location of the sample region relative to the SEM compatible sample container. The stainless steel sheet commercially available from Slon, Kibbutzmergan Michael, Israel, is preferably formed by chemical etching. The sample enclosure forming ring 2113 is preferably applied to the electron beam permeable fluid impermeable membrane 2110 with an adhesive such as catalog number 1193 MVLV, commercially available from Diamix, 51 Greenwood Road, CT06790, USA, for example. It is preferable that they are adhered. Ring 2113 is preferably formed from PMMA (polymethylmethacrylate), such as catalog number 692106001000, commercially available from Ilpen, Barcelona, Spain, and has a height of about 2 mm in a volume of about 20 microliters. Preferably, a sample enclosure is defined. Preferably, the ring 2113 is configured to define a sample enclosure 2114 having an inclined wall configured to form a plurality of radially distributed grooves 2115. The ring 21143 has a peripheral rim 2116 that acts to engage a recess 2117 formed in the protrusion 2118 of the first enclosure element 2100 to steadily seat the ring 2113 in the first enclosure element 2100. It is preferably formed on the outer surface.

    As shown in FIGS. 34A, 34B, 38A, and 38B, a diaphragm 2119 may be integrally formed from an O-ring portion 2120, and an expandable sheet portion 2121 may be sealed in the O-ring portion 2120. Diaphragm 2119 is preferably disposed between inner surface 2126 of second enclosure element 2102 and connecting element 2103. O-ring 2126 is preferably disposed between connecting element 2103 and ring 2113 of subassembly 2108. Diaphragm 2119 and O-ring 2126 eliminate the need for the engagement between elements 2100 and 2102 and connecting element 2103 to be a sealing engagement when enclosure elements 2100 and 2102 and connecting element 2103 are in close engagement. Operates to eliminate and provide its dynamic and static pressure relief.

  The connecting element 2103 preferably has a recess 2128. The connecting element 2103 is formed with a peripheral protrusion 2130 shown in FIGS. 38A and 38B that projects into the recess 2128.

  The positioner 2132 is preferably comprised of two upstanding flexible protrusions 2134, each comprising a ridge 2136 formed at the end 2138 of the protrusion 2134. The positioner 2132 is preferably molded from plastic. As shown in FIGS. 38A and 38B, the protrusions 2134 press against each other when inserted into the recess 2128 of the connection element 2103, and once the ridge 2136 is seated on the protrusion 2130 of the connection element 2103, Snap back to upright position.

  The positioner 2132 is preferably provided with positioning and holding protrusions 2140 extending from the peripheral rim 2142 and extending in the respective radial directions. The positioning and holding projection 2140 is seated in the groove 2115 of the ring 2113 in order to prevent the positioner 2132 from rotating in the ring 2113.

  The coil spring 2144 is disposed on the positioner 2132 between the rim 2142 and the ridge 2136 of the protrusion 2134. The spring 2144 is preferably formed from hardened stainless steel.

  The positioner 2132 and the spring 2144 contact the electron beam transparent and fluid impermeable membrane 2110 to lift the non-liquid sample upward when the enclosure elements 2100 and 2102 and the connecting element 2103 are in tight engagement. Act to move to.

  The first enclosure element 2100 engages the connection element 2103 by connecting a protrusion 2154 formed in the first enclosure element 2100 to a bayonet-type socket 2156 formed in the connection element 2103.

  The projecting portion 2154 includes a first projecting portion 2158 and a second projecting portion 2160, and these projecting portions are formed in a bayonet-type socket 2156, a first recess 2162, and a second recess 2164. Initial engagement of the protrusion 2158 with the recess 2162, for example, loosens the first enclosure element 2100 and the connection element 2103 to prevent accidental damage of the SEM compatible sample container during movement and handling. Provide engagement. Subsequent engagement of the protrusion 2158 with the recess 2164 provides a tight engagement between the enclosure element 2100 and the connection element 2103.

  The second enclosure element 2102 engages the connection element 2103 by connecting a protrusion 2174 formed on the connection element 2103 to a bayonet socket 2176 formed on the second enclosure element 2102.

  The projecting portion 2174 includes a first projecting portion 2178 and a second projecting portion 2180, and these projecting portions are formed in a bayonet-type socket 2176, a first recess 2182, and a second recess 2184. Initial engagement of the protrusion 2178 to the recess 2182, for example, loosens the second enclosure element 2102 and the connection element 2103 to prevent accidental damage of the SEM compatible sample container during movement and handling. Provide engagement. Subsequent engagement of the protrusion 2178 with the recess 2184 provides a tight engagement between the enclosure element 2102 and the connection element 2103.

  The second enclosure element 2102 is preferably formed with a generally central stub 2190 having a through bore 2191 arranged to seat in a suitable recess (not shown) in the specimen stage of a scanning electron microscope. It is a special feature of the present invention that the container shown in FIGS. 34A-43 works in a size that fits the conventional stub recess of a conventional scanning electron microscope and does not need to be deformed whatever it is. is there. It will be appreciated that various forms and sizes of stubs can be provided to fit various scanning electron microscopes.

  Enclosure elements 2100 and 2102 and connecting element 2103 allow the container to be easily seated in a suitable multi-container holder and further allow the user to open and close enclosure elements 2100 and 2102 and connecting element 2103. To assist, it is preferable to provide respective positioning and holding projections 2194, 2196 and 2198 extending radially. Preferably, the relative azimuthal arrangement of the respective protrusions 2194, 2196 and 2198 on each enclosure element 2100 and 2102 and connecting element 2103 is such that these protrusions are shown in FIGS. 37A and 37B. It is preferred that the relative azimuthal alignment between them indicates a tight engagement between the elements.

  Reference is now made to FIGS. 39A, 39B, and 39C, which are three cross-sectional views illustrating the operational aspects of the SEM compatible sample container of FIGS. 34A-38B, respectively, in three stages of operation. FIG. 39A shows the container of FIGS. 34A-38B storing tissue samples 2260 arranged in the configuration shown in FIG. 34B prior to closing enclosure elements 2100 and 2102 and connecting element 2103. FIG. An electron beam transmissive and fluid impermeable membrane 2110 is shown in FIG. 39A to be substantially flat.

  FIG. 39B shows the container of FIG. 39A following full engagement between enclosure elements 2100 and 2102 and connecting element 2103 that creates a seal of tissue sample enclosure 2114 from the surroundings. Note that the tissue sample 2260 is in intimate contact with the electron beam transmissive and fluid impermeable membrane 2110 due to the force exerted by the positioner 2132. It can be seen that the diaphragm 2119 is outward due to pressure buildup in the liquid sample enclosure 2114 as a result of its sealing in this embodiment. Electron beam permeable and fluid impermeable membrane 2110 is outward due to the pressure buildup of tissue sample enclosure 2114 resulting from its sealing in this embodiment, but due to the action of diaphragm 2119. It can be seen that it has only done to a much smaller extent. This can be understood by comparing FIG. 39B to FIG. 29B. Support grid 2112 is shown to be substantially flat.

FIG. 39C shows the container of FIG. 39B when placed in a SEM degassing environment with a vacuum of typically 10 ⁻² to 10 ⁻⁶ mbar. In this environment, the diaphragm 2119 is outward to a greater extent than in the ambient environment of FIG. 39B, and the electron beam permeable and fluid impermeable membrane 2110 and the support grid 2112 provide the ambient environment of FIG. 39B. It can be seen that it is outward to a greater degree than when it is disposed in, but due to the action of diaphragm 2119, it is only to a much lesser degree than in the embodiment of FIG. 29C. . This can be understood by comparing FIG. 39C and FIG. 29C.

  Note that the electron beam transmissive and fluid impermeable membrane 2110 attempts to enter the gap through the gap of the grating 2112 to a greater degree than that generated in the surrounding environment of FIG. 39B. It can be seen that, to a lesser degree than in the embodiment of FIG. 39C. This can be understood by comparing FIG. 39C and FIG. 29C.

  Reference is now made to FIG. 40, which is a simplified and cross-sectional view showing the tissue containing the sample and insertion into the SEM using the SEM compatible sample container of FIGS. 34A-39C. 40 shows the closed container in the configuration of FIG. 34B inserted into the stage 2264 of the SEM 2266. FIG. It will be appreciated that there are SEMs whose container orientation is the opposite of that shown in FIG.

  FIG. 40 is a cross-sectional view showing various operating configurations of the SEM compatible sample container of FIGS. 34A-39B inserted into the SEM using various operating stages and SEM compatible sample containers, Reference is made to FIGS. 41A, 41B, 41C, 41D and 41E.

  FIG. 41A shows a plurality of flexible support elements operating to support particles or cells, such as cell stage 1268, typically placed in incubator 2270. Cell stage 2268 serves to allow the growth of cells on the stage, or alternatively to allow adherence of the growing cells on the stage. Each cell stage 2268 is preferably formed by a porous membrane 2272 surrounded by a ring 2274 and supports a sample 2276 containing cells 2278. Porous membrane 2272 is preferably formed from polyester, such as catalog number 3470, Townswell plate, commercially available from Corning, Acton, USA. Cells 2278 are preferably supported by a porous membrane 2272 on the basal side of cells 2278 in a liquid medium. Porous membrane 2272 serves to maintain the desired level of humidity in the sample during the phase of operation, such as during incubation in incubator 2270, as shown in FIG. 41A.

  It will be appreciated that cells 2278 or particles can be deposited on the flexible support element by adsorbing or filtering the fluid containing the cells or particles through the porous membrane 2272.

  As shown in FIG. 41A, a molecule 2279, such as a cell 2278 molecule or an externally added molecule, is preferably provided on the apical side of the cell 2278. Preferably, the molecule 2279 is linked to a high electron density structure such as a gold colloid that can be visualized with an electron microscope.

  FIG. 41B supports the cells 2276, as specified below, except that it includes a cell stage 2268 mounted on a platform 2282, preferably formed from a non-rigid material, of FIGS. 34A-38B. An SEM compatible sample container 2280 that is identical to the container is shown. Container 2280 is arranged in the configuration shown in FIG. 34B prior to closure of enclosure elements 2100 and 2102 and connecting element 2103. The cell growth platform 2282 is mounted on a suitably configured positioner 2284 corresponding to the positioner 2132 of the example of FIGS. 34A-40. Typically, the cells 2278 grow on the cell stage 2268 in the incubator 2270 while the cell stage 2268 is not mounted on the platform 2282. The mounting of the cell stage 2268 on the platform 2282 and the mounting of the plot form 2282 with the cell stage 2268 on the positioner 2284 are typically performed immediately before the SEM examination is performed.

  FIG. 41C shows container 2280 of FIG. 41B immediately following full engagement between enclosure elements 2100 and 2102 and connection element 2103 that creates a seal of the cell sample enclosure indicated by reference numeral 2286 from the surroundings. Is shown. Note that the sample containing the cells 2278 is in intimate contact with the electron beam permeable and fluid impermeable membrane 2110 due to the force exerted by the positioner 2284. It can be seen that the electron beam transparent and fluid impermeable membrane 1110 is outward due to the pressure build-up of the tissue sample enclosure 2286 resulting from its sealing in this embodiment, but the action of the diaphragm 2119 It can be seen that due to this, it is only to a much lesser extent than in the embodiment of FIG. 31C. This can be understood by comparing FIG. 41C and FIG. 31C. Support grid 2112 is shown to be substantially flat.

FIG. 41D shows the container of FIG. 41C when placed in a SEM degassing environment with a vacuum of typically 10 ⁻² to 10 ⁻⁶ mbar. In this environment, it can be seen that the electron beam permeable and fluid impermeable membrane 2110 and the cell stage 2268 are more outwardly exposed than when placed in the ambient environment of FIG. 41C. Furthermore, the electron beam transmissive and fluid impermeable membrane 2110 attempts to enter the gap through the gap in the grating 2119 to a greater degree than that produced in the ambient environment of FIG. It can be seen that due to the action, it is only to a much lesser extent than in the embodiment of FIG. This can be understood by comparing FIG. 41D and FIG. 31D. In this environment, it can be understood that the grating 2112 is also slightly outward.

  FIG. 41E shows the closed container 2280 in the configuration of FIG. 34B inserted into the stage 2264 of the SEM 2266. It will be appreciated that there are SEMs whose container orientation is opposite to that shown in FIG. 41E.

  Reference is now made to FIG. 42, which is a simplified and cross-sectional view of an SEM inspection of a sample using the SEM compatible sample container of FIGS. 34A-40. As shown in FIG. 42, the container designated by reference numeral 2290 passes through the membrane 2110 (FIGS. 34A-41E) in which the electron beam 2292 generated by the SEM passes through the electron beam transparent and fluid impermeable membrane 2110. , Shown as being placed on stage 2264 of SEM 2266 such that tissue including sample 2294 in container 2290 is irradiated. Backscattered electrons from the sample 2294 pass through an electron beam transparent and fluid impermeable membrane 2110 and are detected by a detector 2296 that forms part of the SEM. One or more additional detectors may be provided, such as secondary electron detector 2298, for example. An x-ray detector (not shown) may be provided for detecting x-ray radiation emitted by the sample 2294 due to electron beam excitation. A cathodoluminescent detector (not shown) may be provided for detecting radiation emitted by the sample 2294 due to electron beam excitation.

  Reference is now additionally made to FIG. 43, which schematically shows details of electron beam interaction with a sample 2294 in a container 2290 according to a preferred embodiment of the present invention. As shown in FIG. 43, the present invention enables high-contrast image formation of feature groups distinguished from each other by their average atomic numbers. In FIG. 43, the cell nucleus 2300, having a relatively high average atomic number, is shown to backscatter electrons more than the surrounding nucleus 2302.

  It should be noted that according to a preferred embodiment of the present invention, imaging inside a sample up to about 2 microns deep can be achieved for electrons having an energy level lower than 50 KeV, as shown in FIG. The cell nuclei 2300 located below the membrane 2110 that is electron beam transparent and fluid impermeable are imaged.

  Reference is now made to FIG. 44, which is a simplified illustration of an SEM-based sample inspection system constructed and operative in accordance with a preferred embodiment of the present invention. As shown in FIG. 44, preferably an automatic positioning system such as a robotic arm as shown is used to transport a number of SEM compatible sample containers 2602 through the system. It will be appreciated that manual intervention may be used as appropriate in one or more stages.

  Thereafter, the individual containers 2602 are placed on the electron microscope specimen stage 2610 and then introduced into the scanning electron microscope 2612. The resulting image is visually inspected by an operator and / or analyzed by conventional image analysis functions typically implemented in computer 2614.

  Reference is now made to FIG. 45, which is a simple cross-sectional view of sample inspection within an inverted optical microscope using the sample holder 3000. The sample holder 3000 is preferably defined by a light transmissive film 3002 and supports the sample 3004 in a liquid medium. The film 3002 acts to allow light to pass through it and strike the sample 3004. It will be appreciated that the sample may include a fluid or a particulate component such as, for example, cell 3006.

  As shown in FIG. 45, the sample holder 3000 is shown in a state of being supported by a support ring 3010 disposed in the specimen stage 3011 of the inverted optical microscope 3012. An immersion fluid 3014, such as oil or gel, having a refractive index similar to that of the objective lens 3016 of the inverted optical microscope 3012 is placed between the membrane 3002 and the objective lens 3016 to provide optimal optical properties. be able to.

  The sample holder 3000 with the membrane 3002, preferably less than 10 microns thick, is lighter due to the shorter distance from the lens to the sample and the weaker optical activity of the membrane 3002 when compared to conventional glass slides. It will be appreciated that improved imaging can be provided by a variety of methods including field of view, dark field, confocal microscopy, and total internal reflection fluorescence microscopy.

  Reference is now made to FIG. 46 illustrating a methodology for an electron microscope that reduces or prevents damage to the sample or sample container due to electron beam irradiation. Samples, particularly organic or polymer samples, are known to suffer damage due to electron beam irradiation. Applicants have noted that the electron beam damage to the film of the sample holder described above with reference to FIGS. 1A-45 can occur due to electron beam irradiation on the film and, alternatively, or Additionally, it has been recognized that this can occur due to electron beam irradiation on the sample, which generates reactive species that can attack the membrane.

  A particular feature of the present invention is that a methodology for preventing or reducing electron beam induced damage provides a sample of organic or polymeric material that is sensitive to electron beam irradiation induced damage, wherein the sample includes an electron beam That is, a protective material that at least partially prevents irradiation-induced damage is added, and each step of irradiating the sample and the protective material with an electron beam in an electron microscope is provided.

  Also, a special feature of the present invention is that a methodology for preventing or reducing electron beam induced damage places a sample in a sample enclosure, the sample enclosure being sensitive to electron beam irradiation induced damage. A permeable, fluid impermeable membrane, and further comprising a surrounding enclosure sealed to the membrane and forming the sample enclosure with the membrane, the sample being subjected to electron beam radiation induced damage to the membrane It also means that a step of adding a protective material that at least partially prevents the sample and irradiating the sample and the protective material with an electron beam in an electron microscope is provided.

It will be appreciated that the film may be sensitive to electron beam induced damage resulting from electron beam irradiation of the film or electron beam irradiation of the sample.
Referring now to FIG. 46, there is shown a simple view and cross-sectional view of a sample SEM inspection using the SEM compatible sample container of FIGS. 11A-15B. It will be appreciated that the protection methodology described herein with reference to FIG. 46 is not limited to SEM microscopes and is equally applicable to other types of electron microscopes such as, for example, TEM microscopes. .

  As shown in FIG. 46, the container designated by reference numeral 3500 has an electron beam 3506 generated by the SEM identical to the electron beam transmissive and fluid impermeable membrane shown in FIGS. 1A-44. Is shown as being placed on stage 3502 of SEM 3504 so as to pass through an electron beam transmissive and fluid impermeable membrane 3510 and be irradiated to a liquid containing sample 3512 in container 3500. . Sample 3512 typically includes an organic or polymeric material. Backscattered electrons from the sample 3512 pass through an electron beam transparent and fluid impermeable membrane 3510 and are detected by a detector 3514 that forms part of the SEM. For example, one or more additional detectors such as secondary electron detector 3516 may be provided. An x-ray detector (not shown) may be provided for detecting x-ray radiation emitted by the sample 3512 due to electron beam excitation. A cathodoluminescent detector (not shown) may be provided for detecting radiation emitted by the sample 3512 due to electron beam excitation.

  As symbolically shown in FIG. 46, irradiation of the electron beam sample 3512 and / or film 3510 may generate reactive species such as ions or free radicals, for example. These reactive species can cause damage to sample 3512 and / or membrane 3510.

  In accordance with a preferred embodiment of the present invention, protective material is added to sample 3512 to at least partially deactivate the reactive species so as to prevent damage to sample 3512 and / or membrane 3510. Examples of suitable protective materials include 4- (2-hydroxyl) -1-piperazineethanesulfonic acid (HEPES-KOH), (25 mM, pH 7.5); trishydroxymethylaminomethane (Tris-HCl), 10 mM, pH 7.5; D glucose, 25 mM; D-sorbitol, 25 mM; L-ascorbic acid, 50 mM; L-carnosine, 50 mM; nicotinamide adenine dinucleotide (NADH), 25 mM; and fetal bovine serum, 10% (v / v) is included.

  As symbolically shown in FIG. 46, the molecules of the protective material indicated by reference material 3520 attempt to react with the reactive species indicated by reference numeral 3522, thus rendering them inactive, and sample 3512 And / or reduce or prevent damage to the membrane 3510. If this is not done, the damage will occur as a result of irradiating the sample 3512 or film 3510 with the electron beam 3506.

  It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described hereinabove. The scope of the present invention includes both the various feature combinations and sub-combinations described above, as well as variations and modifications thereof, as would occur to those skilled in the art upon reading the foregoing specification and do not exist in the prior art. It is covered.

1A and 1B are simplified exploded views, each directed to the opposite side, of a disassembled SEM compatible sample container constructed and operative in accordance with a preferred embodiment of the present invention. 2A and 2B are partial cross-sectional views of simplified partial views of the subassemblies of the container of FIGS. 1A and 1B, each directed to the opposite side. 3A and 3B are simplified exploded views, each directed to the opposite side, showing the SEM compatible sample container of FIGS. 1A-2B partially assembled. 4A and 4B are simplified views, each directed to the opposite side, showing the fully assembled SEM compatible sample container of FIGS. 1A-3B. 5A and 5B are partial cross-sectional views of the simplified partial views, taken along the lines VA-VA and VB-VB, respectively, facing the opposite sides in FIGS. 3A and 3B, respectively. FIGS. 6A, 6B and 6C are three cross-sectional views illustrating the operating mode of the SEM-compatible sample container of FIGS. 1A-5B, respectively, in three stages of operation. 7A, 7B, 7C, 7D and 7E are simplified cross-sectional views of cell growth, liquid removal, liquid addition, sealing and insertion into the SEM using the SEM compatible sample containers of FIGS. 1A-6C. is there. FIGS. 8A, 8B and 8C are simplified cross-sectional views of sample-containing liquid, sealed and inserted into the SEM using the SEM compatible sample container of FIGS. 1A-6C. FIG. 9 is a simplified and cross-sectional view of a SEM inspection of a sample using the SEM compatible sample container of FIGS. 1A-6C. FIG. 10 is a simplified diagram showing an enlarged SEM inspection state of the sample in the situation of FIG. 11A and 11B are simplified exploded views, each directed to the opposite side, of a disassembled SEM compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention. 12A and 12B are partial cross-sectional views of simplified partial views of the subassemblies of the container of FIGS. 11A and 11B, each directed to the opposite side. 13A and 13B are simplified exploded views, each directed to the opposite side, showing the SEM compatible sample container of FIGS. 11A-12B partially assembled. 14A and 14B are simplified views, each directed to the opposite side, showing the fully assembled SEM compatible sample container of FIGS. 11A-13B. FIGS. 15A and 15B are partial cross-sectional views of the simplified partial views, taken along lines XVA-XVA and XVB-XVB, respectively, in FIGS. 13A and 13B, each directed to the opposite side. FIGS. 16A, 16B, and 16C are three cross-sectional views illustrating the operating mode of the SEM compatible sample container of FIGS. 11A-15B, respectively, in three stages of operation. 17A, 17B, 17C, 17D and 17E are simplified cross-sectional views of cell growth, liquid removal, liquid addition, sealing and insertion into the SEM using the SEM compatible sample containers of FIGS. 11A-16C. is there. 18A, 18B, and 18C are simplified cross-sectional views of sample-containing liquid, sealed, and inserted into the SEM using the SEM-compatible sample containers of FIGS. 11A-16C. FIG. 19 is a simplified and cross-sectional view of a sample SEM inspection using the SEM compatible sample container of FIGS. 11A-16C. FIG. 20 is a simplified diagram showing an enlarged SEM inspection state of the sample in the situation of FIG. 21A and 21B are simplified exploded views of a multiple sample holder prior to microscopy for use with the SEM compatible sample container of the type shown in FIGS. 1A-20. 22A, 22B, and 22C are simplified views of the multiple sample holder prior to microscopy of FIGS. 21A-21B with a suction device and pipette. FIG. 23 is a simplified diagram of an SEM-based sample inspection system constructed and operative in accordance with a preferred embodiment of the present invention. 24A and 24B are simplified exploded views, each directed to the opposite side, of a disassembled SEM compatible sample container constructed and operative in accordance with a preferred embodiment of the present invention. FIGS. 25A and 25B are partial cross-sectional views of simplified partial views of the subassemblies of the container of FIGS. 24A and 24B, each directed to the opposite side. 26A and 26B are simplified exploded views, each directed to the opposite side, showing the SEM compatible sample container of FIGS. 24A-25B partially assembled. 27A and 27B are simplified views, each directed to the opposite side, showing the fully assembled SEM compatible sample container of FIGS. 24A-26B. FIGS. 28A and 28B are partial cross-sectional views of FIGS. 26A and 26B, taken along lines XXVIIIA-XXVIIIA and line XXVIIIB-XXVIIIB, respectively, and directed to the opposite sides. FIGS. 29A, 29B and 29C are three cross-sectional views illustrating the operating mode of the SEM compatible sample container of FIGS. 24A-28B, respectively, in three stages of operation. FIG. 30 is a simplified and cross-sectional view showing tissue containing the sample and insertion into the SEM using the SEM compatible sample container of FIGS. 24A-29C. FIGS. 31A, 31B, 31C, 31D and 31E illustrate the various stages of operation and insertion into the SEM, respectively, using an SEM compatible sample container constructed and operated in accordance with another preferred embodiment of the present invention. FIG. 6 is a simplified cross-sectional view of an operating mode of a compatible sample container. FIG. 32 is a simplified and cross-sectional view of an SEM inspection of a sample using the SEM compatible sample container of FIGS. 24A-30. FIG. 33 is a simplified diagram showing an enlarged SEM inspection state of the sample in the situation of FIG. 34A and 34B are simplified exploded views, each directed to the opposite side, of a disassembled SEM compatible sample container constructed and operative in accordance with another preferred embodiment of the present invention. FIGS. 35A and 35B are partial cross-sectional views of simplified partial views of the subassemblies of the container of FIGS. 34A and 34B, each directed to the opposite side. 36A and 36B are simplified exploded views, each directed to the opposite side, showing the SEM compatible sample container of FIGS. 34A-35B partially assembled. FIGS. 37A and 37B are simplified views, each directed to the opposite side, showing the fully assembled SEM compatible sample container of FIGS. 34A-36B. FIGS. 38A and 38B are partial cross-sectional views of simplified partial views, taken along lines XXXVIIIA-XXXVIIIA and line XXXVIIIB-XXXVIIIB, respectively, in FIGS. 36A and 36B, each facing the opposite side. FIGS. 39A, 39B and 39C are three cross-sectional views illustrating the operating mode of the SEM-compatible sample container of FIGS. 34A-38B, respectively, in three stages of operation. FIG. 40 is a simplified cross-sectional view and a simplified view of each state of tissue containing the sample and insertion into the SEM using the SEM compatible sample container of FIGS. 35A-39C. 41A, 41B, 41C, 41D, and 48E are SEMs showing various stages of operation and insertion into the SEM, respectively, using an SEM compatible sample container constructed and operated in accordance with another preferred embodiment of the present invention. FIG. 6 is a simplified cross-sectional view of an operating mode of a compatible sample container. 42 is a simplified and cross-sectional view of a SEM inspection of a sample using the SEM compatible sample container of FIGS. 34A-39C. FIG. 43 is a simplified enlarged view of the SEM inspection state of the sample in the situation of FIG. FIG. 44 is a simplified diagram of an SEM-based sample inspection system constructed and operative in accordance with a preferred embodiment of the present invention. FIG. 45 is a simplified partial and partial cross-sectional view of a sample in an inverted optical microscope constructed and operative in accordance with a preferred embodiment of the present invention. 46 is a simplified diagram and cross-sectional view of a sample SEM inspection using the SEM compatible sample container of FIGS. 11A-20.

Claims (130)

A SEM compatible sample container,
A sample enclosure comprising: an electron beam permeable fluid impermeable membrane; and a surrounding enclosure sealed to the membrane to form the sample enclosure with the membrane;
A closure of the sample enclosure having a quick connect attachment function for sealing engagement with the sample enclosure;
A SEM compatible sample container.   The SEM-compatible sample container according to claim 1, wherein the quick-connect attachment function comprises a bayonet-type connection function.   The SEM compatible sample container of claim 2, wherein the surrounding enclosure is at least partially conductive.   The SEM compatible sample container of claim 2 or 3, further comprising a pressure relief diaphragm associated with the sample enclosure.   The SEM compatible sample container of any one of claims 1 to 4, further comprising at least one reference coordination indicator associated with the membrane.   6. The SEM compatible sample container of any one of claims 1 to 5, further comprising at least one membrane support grid that supports the membrane and has a reference coordination indicator.   The membrane is polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, "Perrodion", "Colodion", "Kapton", "Form Bar", "Vinyleck", "Bat Bar", "Pioroform", "Parilene" The SEM-compatible sample container of any one of claims 2 to 6, wherein the sample container is formed from a material selected from the group consisting of carbon dioxide, silicon dioxide, silicon monoxide, and carbon.   The sample enclosure is pre-assembled and prepared to receive a liquid containing a sample, following which the closure of the sample enclosure can be easily hermetically coupled using the quick connect attachment feature. The SEM compatible sample container according to any one of claims 2 to 6, which is capable. A SEM compatible sample container comprising a liquid sample enclosure,
The liquid sample enclosure comprises an electron beam permeable and fluid impermeable membrane and a surrounding enclosure sealed to the membrane to form the liquid sample enclosure with the membrane;
The SEM compatible sample container, wherein the sample enclosure can contain liquid at a depth that is not permeable by electrons having an energy level of less than 50 KeV.   The SEM compatible sample container of claim 9, further comprising a liquid sample enclosure closure having a quick connect attachment feature for sealing engagement with the liquid sample enclosure.   11. The SEM compatible sample container of claim 10, wherein the quick connect attachment function comprises a bayonet connection function.   The SEM compatible sample container of claim 11, wherein the surrounding enclosure is at least partially conductive.   The SEM-compatible sample container of claim 11 or 12, further comprising a pressure relief diaphragm associated with the sample enclosure.   The SEM compatible sample container of any one of claims 9 to 13, further comprising at least one reference coordination indicator associated with the membrane.   The SEM-compatible sample container of any one of claims 9 to 14, further comprising at least one membrane support grid that supports the membrane and has a reference coordination indicator.   The membrane is polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, "Perrodion", "Colodion", "Kapton", "Form Bar", "Vinyleck", "Bat Bar", "Pioroform", "Parilene" The SEM-compatible sample container of any one of claims 11 to 15, wherein the sample container is formed from a material selected from the group consisting of: silicon dioxide, silicon monoxide, and carbon.   The sample enclosure is pre-assembled and prepared to receive a liquid containing a sample, following which the closure of the sample enclosure can be easily hermetically coupled using the quick connect attachment feature. 15. A SEM compatible sample container according to any one of claims 11 to 14, which is capable. A SEM compatible sample container,
A sample pan portion comprising: an electron beam permeable fluid impermeable membrane; and a surrounding enclosure sealed to the membrane to form the sample pan portion with the membrane; and
An outer enclosure arranged around the sample pan and forming an aperture for passing electrons through the membrane with the interior of the pan;
A SEM compatible sample container.   The SEM compatible sample container of claim 18, wherein the sample pan can contain a liquid at a depth that is not permeable by electrons having an energy level of less than 50 KeV.   20. The SEM compatible sample container of claim 18 or 19, further comprising an outer enclosure closure having a quick connect attachment feature for sealing engagement with the surrounding enclosure.   21. The SEM compatible sample container of claim 20, wherein the quick connect attachment function comprises a bayonet connection function.   The SEM compatible sample container of claim 21, wherein the surrounding enclosure is at least partially conductive.   23. The SEM compatible sample container of claim 21 or 22, further comprising a pressure relief diaphragm associated with the sample pan.   24. A SEM compatible sample container according to any one of claims 18 to 23, further comprising at least one reference coordination indicator associated with the membrane.   25. A SEM compatible sample container according to any one of claims 18 to 24, further comprising at least one membrane support grid supporting the membrane and having a reference coordination indicator.   The membrane is polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, "Perrodion", "Colodion", "Kapton", "Form Bar", "Vinyleck", "Bat Bar", "Pioroform", "Parilene" 26. A SEM compatible sample container according to any one of claims 21 to 25, formed from a material selected from the group consisting of: silicon dioxide, silicon monoxide, and carbon.   The sample enclosure is pre-assembled and prepared to receive the liquid containing the sample, and following the preparation, the outer enclosure closure can be easily hermetically connected using the quick connect attachment feature. 24. A SEM compatible sample container according to any one of claims 21 to 23. A SEM compatible sample container,
An enclosure that forms an aperture for electrons to pass through;
A sample pan disposed within the enclosure and comprising an electron beam permeable and fluid impermeable membrane;
With
The SEM-compatible sample container, wherein the aperture is positioned relative to the membrane to communicate electrons with the interior of the enclosure through the membrane.   29. The SEM compatible sample container of claim 28, wherein the sample pan is formed by the membrane together with the enclosure.   30. The SEM compatible sample container of claim 29, wherein the sample pan is formed by the membrane with a separate pan wall disposed within the enclosure.   32. The SEM compatible sample container of claim 30, wherein the separate dish wall is sealed to the membrane.   32. A SEM-compatible sample container according to any one of claims 28 to 31, wherein the sample pan can contain liquid at a depth that is not permeable by electrons having an energy level of less than 50 KeV.   33. The SEM compatible sample container of any one of claims 28 to 32, further comprising a closure having a quick connect attachment feature for sealing engagement with the enclosure.   34. The SEM compatible sample container of claim 33, wherein the quick connect attachment function comprises a bayonet connection function.   35. The SEM compatible sample container of claim 34, wherein the enclosure is at least partially conductive.   36. The SEM compatible sample container of claim 34 or 35, further comprising a pressure relief diaphragm associated with the sample pan.   37. A SEM compatible sample container according to any one of claims 28 to 36, further comprising at least one reference coordination indicator associated with the membrane.   38. The SEM compatible sample container of any one of claims 28 to 37, further comprising at least one membrane support grid that supports the membrane and has a reference coordination indicator.   The membrane is polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, "Perrodion", "Colodion", "Kapton", "Form Bar", "Vinyleck", "Bat Bar", "Pioroform", "Parilene" 39. The SEM compatible sample container of any one of claims 34 to 38, formed from a material selected from the group consisting of: silicon dioxide, silicon monoxide, and carbon.   The sample enclosure is pre-assembled and prepared to receive a liquid containing a sample, following which the closure can be easily hermetically coupled using the quick connect attachment feature. Item 36. The SEM compatible sample container according to Item 34 or 35. A SEM compatible sample container,
A sample pan assembly forming an aperture, the aperture communicating electrons through the aperture, wherein the sample pan assembly is an electron beam transmissive and fluid impermeable membrane that at least partially forms a sample enclosure Comprising the sample dish assembly; and
A sample positioner configured to have a linear non-rotating motion in engagement with the sample, thereby positioning the sample adjacent to the membrane;
A closure having a quick connect attachment function for sealing engagement with the enclosure;
With
The aperture is positioned relative to the membrane such that electrons are in communication through the aperture with the sample adjacent to the membrane, the aperture penetrating the membrane, an SEM compatible sample container.   42. The SEM compatible sample container of claim 41, wherein the sample positioner comprises a flexible support element.   43. The SEM compatible sample container of claim 42, wherein the flexible support element comprises a cell support element.   44. The SEM compatible sample container of claim 42 or 43, wherein the flexible support element comprises a cell growth element.   45. A SEM compatible sample container according to any one of claims 42 to 44, wherein the flexible support element comprises a fluid filter element.   46. A SEM compatible sample container according to any one of claims 42 to 45, wherein the flexible support element comprises a membrane.   47. A SEM compatible sample container according to any one of claims 42 to 46, wherein the flexible support element comprises an elastic membrane support.   48. A SEM compatible sample container according to any of claims 42 to 47, wherein the flexible support element is at least partially permeable to liquid.   49. A SEM compatible sample container according to any one of claims 42 to 48, wherein the function of the quick connect attachment comprises a bayonet connection function.   50. A SEM compatible sample container according to any one of claims 42 to 49, wherein the sample enclosure is at least partially conductive.   51. A SEM compatible sample container according to any one of claims 42 to 50, wherein the sample positioner comprises a spring.   52. A SEM compatible sample container according to any one of claims 42 to 51, further comprising a pressure relief diaphragm associated with the sample pan assembly.   53. The SEM compatible sample container of any one of claims 41 to 52, further comprising at least one reference coordination indicator associated with the membrane.   54. The SEM compatible sample container of any one of claims 41 to 53, further comprising at least one membrane support grid that supports the membrane and has a reference coordination indicator function.   The membrane is polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, "Perrodion", "Colodion", "Kapton", "Form Bar", "Vinyleck", "Bat Bar", "Pioroform", "Parilene" 55. The SEM compatible sample container of any one of claims 42 to 54, formed from a material selected from the group consisting of: silicon dioxide, silicon monoxide, and carbon.   The sample enclosure is pre-assembled and prepared to receive a liquid containing a sample, following which the closure can be easily hermetically coupled using the quick connect attachment feature. Item 45. The SEM compatible sample container according to any one of Items 42 to 44. A SEM compatible sample container,
A sample pan assembly forming an aperture, the aperture communicating electrons through the aperture, wherein the sample pan assembly is an electron beam transmissive and fluid impermeable membrane that at least partially forms a sample enclosure Comprising the sample dish assembly; and
A pressure relief diaphragm associated with the sample pan assembly;
A SEM compatible sample container.   58. The SEM compatible sample container of claim 57, wherein the pressure relief diaphragm is disposed within the sample enclosure.   59. The SEM compatible sample container of claim 57 or 58, further comprising a closure having a quick connect attachment feature for sealing engagement with the sample enclosure.     60. The SEM compatible sample container of claim 59, wherein the quick connect attachment function comprises a bayonet connection function.   61. The SEM compatible sample container of claim 60, wherein the sample enclosure is at least partially conductive.   62. The SEM compatible sample container of any one of claims 57 to 61, further comprising at least one reference coordination indicator associated with the membrane.   63. The SEM compatible sample container of any one of claims 57 to 62, further comprising at least one membrane support grid that supports the membrane and has a reference coordination indicator function.   The membrane is made of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, "Perrodion", "Colodion", "Kapton", "Form Bar", "Vinyleck", "Bat Bar", "Pioroform", "Parilene" 64. The SEM compatible sample container of any one of claims 60 to 63, wherein the SEM compatible sample container is formed from a material selected from the group consisting of: silicon dioxide, silicon monoxide, and carbon.   The sample enclosure is pre-assembled and prepared to receive a liquid containing a sample, following which the closure can be easily hermetically coupled using the quick connect attachment feature. Item 65. The SEM-compatible sample container according to any one of Items 60 to 64. A SEM compatible sample container,
A sample pan assembly forming an aperture, the aperture communicating electrons through the aperture, wherein the sample pan assembly is an electron beam transmissive and fluid impermeable membrane that at least partially forms a sample enclosure Comprising the sample dish assembly; and
At least one reference coordination indicator associated with the membrane;
A SEM compatible sample container.   68. The SEM compatible sample container of claim 66, further comprising at least one membrane support grid that supports the membrane and has a reference coordination indicator function.   68. The SEM compatible sample container of claim 66 or 67, further comprising a closure having a quick connect attachment feature for sealing engagement with the sample enclosure.   69. The SEM compatible sample container of claim 68, wherein the quick connect attachment function comprises a bayonet connection function.   70. A SEM compatible sample container according to any one of claims 66 to 69, wherein the sample enclosure is at least partially conductive.   71. The SEM compatible sample container of any one of claims 66 to 70, further comprising a pressure relief diaphragm associated with the sample pan assembly.   The membrane is made of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, "Perrodion", "Colodion", "Kapton", "Form Bar", "Vinyleck", "Bat Bar", "Pioroform", "Parilene" 70. A SEM compatible sample container according to any one of claims 66 to 69, formed from a material selected from the group consisting of: silicon dioxide, silicon monoxide, and carbon.   The sample enclosure is pre-assembled and prepared to receive a liquid containing a sample, following which the closure can be easily hermetically coupled using the quick connect attachment feature. Item 71. The SEM compatible sample container according to any one of Items 68 to 70. A multi-sample container system prior to microscopy with SEM compatibility,
A plurality of SEM compatible sample containers according to any one of claims 1 to 73;
A support for supporting the plurality of SEM compatible sample containers;
A multi-sample container system.   The support includes a light transmission portion disposed below at least one of the membranes in the plurality of SEM compatible sample containers, whereby the plurality of SEM compatible sample containers are supported by the support. 75. The multiple sample container system of claim 74, wherein an inspection by a light microscope can be performed on a sample in at least one of the SEM compatible sample containers during.   76. The multiple sample container system of claim 74 or 75, further comprising a cover arranged to cover the support and the plurality of SEM compatible sample containers supported by the support.   The support holds a liquid that is useful for maintaining the humidity of the sample within the plurality of SEM compatible sample containers while the plurality of SEM compatible sample containers are supported by the support. 77. A multiple sample container system according to any one of claims 74 to 76, comprising at least one liquid reservoir for the purpose.   78. A multiple sample container system according to any one of claims 74 to 77, wherein the SEM compatible multiple sample container is provided with a suction device and a pipette.   79. The suction device according to claim 78, wherein the suction device is configured to prevent its physical engagement with the membrane of the plurality of SEM compatible sample containers upon its operative engagement with the support. Multiple sample container system as described.   80. A multiple sample container system according to claim 78 or 79, wherein the pipette is provided with a collar element to prevent unintentional engagement of the pipette with the membrane.   81. The multiple sample container system of any one of claims 74 to 80, wherein the multiple sample container prior to microscopic examination is dimensioned to be compatible with conventional cell biology equipment.   The multiple sample container system according to any one of claims 74 to 81, wherein the support part has a holding function for detachably holding individual containers of the plurality of SEM compatible sample containers with respect to each other. . A SEM system,
SEM,
A sample pan assembly forming an aperture, the aperture communicating electrons through the aperture, wherein the sample pan assembly is an electron beam transmissive and fluid impermeable membrane that at least partially forms a sample enclosure Comprising the sample dish assembly; and
An X-ray detector arranged to receive X-rays during a SEM examination from a sample-containing liquid arranged in the sample enclosure;
An SEM system comprising:   84. The SEM system of claim 83, further comprising a sample enclosure closure having a quick connect attachment feature for sealing engagement with the sample enclosure.   85. The SEM system of claim 84, wherein the quick connect attachment function comprises a bayonet connection function.   86. The SEM system of claim 85, wherein the sample enclosure is at least partially conductive.   87. The SEM system of claim 85 or 86, further comprising a pressure relief diaphragm associated with the sample enclosure.   88. The SEM system according to any one of claims 83 to 87, further comprising at least one reference coordination indicator associated with the membrane.   89. The SEM system of any one of claims 83 to 88, further comprising at least one membrane support grid that supports the membrane and has a reference coordination indicator function.   The membrane is made of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, "Perrodion", "Colodion", "Kapton", "Form Bar", "Vinyleck", "Bat Bar", "Pioroform", "Parilene" 90. The SEM system of any one of claims 85 to 89, formed from a material selected from the group consisting of: silicon dioxide, silicon monoxide, and carbon.   The sample enclosure is pre-assembled and prepared to receive the liquid containing the sample, and following the preparation, the sample enclosure closure can be easily hermetically coupled using the quick connect attachment feature. The SEM system according to any one of claims 85 to 90. A SEM system,
SEM,
A SEM-compatible sample container having a sample enclosure, the sample enclosure comprising an electron beam permeable fluid impermeable membrane and a surrounding enclosure sealed to the membrane to form the liquid sample enclosure with the membrane And the SEM compatible sample container comprising:
A closure of the sample enclosure having a quick connect attachment function for sealing engagement with the sample enclosure;
An SEM system comprising:   94. The SEM system of claim 92, wherein the quick connect attachment function comprises a bayonet connection function.   94. The SEM system of claim 93, wherein the surrounding enclosure is at least partially conductive.   95. The SEM system of claim 93 or 94, further comprising a pressure relief diaphragm associated with the sample enclosure.   96. The SEM system of any one of claims 92 to 95, further comprising at least one reference coordination indicator associated with the membrane.   97. The SEM system according to any one of claims 92 to 96, further comprising at least one membrane support grid that supports the membrane and has a reference coordination indicator function.   The membrane is made of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, "Perrodion", "Colodion", "Kapton", "Form Bar", "Vinyleck", "Bat Bar", "Pioroform", "Parilene" 98. The SEM system according to any one of claims 93 to 97, wherein the SEM system is formed from a material selected from the group consisting of silicon dioxide, silicon monoxide, and carbon.   The sample enclosure is pre-assembled and prepared to receive the liquid containing the sample, and following the preparation, the sample enclosure closure can be easily hermetically coupled using the quick connect attachment feature. 99. The SEM system according to any one of claims 93 to 98. A method for performing an inspection with a scanning electron microscope,
Placing a sample in a sample enclosure, the sample enclosure comprising an electron beam permeable fluid impermeable membrane and a surrounding enclosure sealed to the membrane to form the sample enclosure with the membrane; A closure of the sample enclosure having a quick connect attachment function for sealing engagement with the sample enclosure; and
Sealing the sample enclosure with the sample enclosure closure;
Placing the sample enclosure in an electron beam;
Analyzing the result of the interaction between the electron beam and the sample;
A method comprising:   101. The method for performing a scanning electron microscope inspection according to claim 100, further comprising removing liquid from the sample enclosure prior to the sealing step.   102. The method for performing a scanning electron microscope examination according to claim 100 or 101, further comprising the step of adding liquid to the sample enclosure prior to the sealing step.   103. A method for performing an inspection with a scanning electron microscope according to any one of claims 100 to 102, comprising the step of culturing the sample in the sample enclosure. Analysis of the result of the interaction between the sample and the electron beam is
X-ray detection and detection of light in the ultraviolet to infrared range;
Detection of backscattered electrons,
104. A method for performing an inspection with a scanning electron microscope according to any one of claims 100 to 103, performed by at least one of detecting secondary electrons. A method for performing an inspection with a scanning electron microscope,
Placing a sample in a sample enclosure, the sample enclosure comprising an electron beam permeable fluid impermeable membrane and a surrounding enclosure sealed to the membrane to form the sample enclosure with the membrane; A closure of the sample enclosure having a quick connect attachment function for sealing engagement with the sample enclosure; and
Placing a sample positioner arranged to position the sample adjacent to the membrane;
Sealing the sample enclosure with a closure of the sample enclosure;
Placing the sample enclosure in an electron beam;
A method comprising the steps of analyzing the results of the interaction between the electron beam and the sample.   106. The method for performing a scanning electron microscope examination according to claim 105, further comprising removing liquid from the sample enclosure prior to the sealing step.   107. A method for performing a scanning electron microscope examination as claimed in claim 105 or 106, further comprising adding liquid to the sample enclosure prior to the sealing step.   108. A method for performing an inspection with a scanning electron microscope according to any one of claims 105 to 107, further comprising culturing the sample in the sample enclosure. Analysis of the result of the interaction between the sample and the electron beam is
X-ray detection and detection of light in the ultraviolet to infrared range;
Detection of backscattered electrons,
109. A method for performing an inspection with a scanning electron microscope according to any one of claims 105 to 108, performed by at least one of detecting secondary electrons.   110. A method for performing a scanning electron microscope examination according to any one of claims 105 to 109, wherein the sample positioner comprises a flexible support element.   111. A method for performing a scanning electron microscope examination as in claim 110, wherein the flexible support element comprises a cell support element.   112. A method for performing a scanning electron microscope examination according to claim 110 or 111, wherein the flexible support element comprises a cell growth element.   113. A method for performing a scanning electron microscope examination as claimed in any one of claims 110 to 112, wherein the flexible support element comprises a fluid filter element.   114. A method for performing a scanning electron microscope examination as claimed in any one of claims 110 to 113, wherein the flexible support element comprises a membrane.   115. A method for performing an inspection with a scanning electron microscope according to any one of claims 110 to 114, wherein the flexible support element comprises an elastic membrane support.   116. A method for performing a scanning electron microscope examination as claimed in any one of claims 110 to 115, wherein the flexible support element is at least partially permeable to liquid.   117. The method for performing an inspection with a scanning electron microscope according to any one of claims 105 to 116, wherein the function of the quick connection attachment comprises a bayonet connection function.   118. The method for performing a scanning electron microscope examination as in claim 117, wherein the sample enclosure is at least partially conductive.   119. A method for performing a scanning electron microscope examination as claimed in claim 117 or 118, wherein the sample positioner comprises a spring.   120. The method for performing an inspection with a scanning electron microscope according to any one of claims 117 to 119, further comprising a pressure relief diaphragm associated with the sample pan assembly.   121. A method for performing a scanning electron microscope examination as claimed in any one of claims 105 to 120, further comprising at least one reference coordination indicator associated with the membrane.   The scanning electron microscope according to any one of claims 105 to 121, further comprising at least one film support grid supporting the film and having a reference coordination indicator function. Method.   The membrane is made of polyimide, polyamide, polyamide-imide, polyethylene, polypyrrole, “Perrodion”, “Colodion”, “Kapton”, “Form Bar”, “Vinyleck”, “Bat Bar”, “Pioloform”, “Parilene” 123. A method for performing an inspection with a scanning electron microscope according to any one of claims 110 to 122, wherein the method is formed from a material selected from the group consisting of: silicon dioxide, silicon monoxide, and carbon. .   The sample enclosure is pre-assembled and prepared to receive a liquid containing a sample, following which the closure can be easily hermetically coupled using the quick connect attachment feature. 120. A method for performing an inspection using a scanning electron microscope according to any one of items 110 to 123. A method using an electron microscope,
Providing a sample of organic or polymeric material that is sensitive to damage induced by electron beam irradiation;
Adding a protective material to the sample that at least partially prevents damage induced by the electron beam irradiation;
A method comprising the steps of irradiating the sample and the protective material with an electron beam in an electron microscope. Providing the sample comprises:
Arranging the sample in a sample enclosure;
The sample enclosure is
129. The method of claim 125, comprising an electron beam permeable fluid impermeable membrane and a surrounding enclosure sealed to the membrane to form the sample enclosure with the membrane. A method using an electron microscope,
Placing a sample in a sample enclosure, the sample enclosure comprising an electron beam permeable and fluid impermeable membrane that is sensitive to damage induced by electron beam irradiation and sealed to the membrane A surrounding enclosure that forms the sample enclosure with a membrane; and
Adding a protective material to the sample that at least partially prevents damage induced by the electron beam irradiation;
Irradiating the sample and the protective material with an electron beam in an electron microscope;
A method comprising:   128. The method of claim 127, wherein the film is sensitive to electron beam irradiation induced damage due to electron beam irradiation of the film.   129. The method of claim 127 or 128, wherein the film is sensitive to electron beam irradiation induced damage resulting from electron beam irradiation of the sample. A top open microscope sample container,
A light transmissive and fluid impermeable sample support of thickness less than 10 microns;
An open top ambient enclosure that is sealed to the sample support and forms a top open microscope sample container with the sample support;
A top open microscope sample container comprising:

Images