Patent Publication Number: US-9431213-B2

Title: Scanning electron microscope, an interface and a method for observing an object within a non-vacuum environment

Description:
RELATED APPLICATIONS 
     This application is a continuation of U.S. Pat. application Ser. No. 13/663,684, filed on Oct. 30, 2012, entitled “SCANNING ELECTRON MICROSCOPE, AN INTERFACE AND A METHOD FOR OBSERVERING AN OBJECT WITHIN A NON-VACUUM ENVIROMENT”, which is a continuation of U.S. Pat. application Ser. No. 13/002,448, filed Mar. 10, 2011, now U.S. Pat. No. 8,334,510, entitled “SCANNING ELECTRON MICROSCOPE, AN INTERFACE AND A METHOD FOR OBSERVING AN OBJECT WITHIN A NON-VACCUM ENVIRONMENT”, which is a U.S. National Phase application of PCT application No. PCT/IL2009/000660, filed Jul. 2, 2009which claims priority of U.S. Provisional Pat. application Ser. Nos. 61/077,970; 61/077,977; 61/077,981 and 61/077,955, all of which were filed on Jul. 3, 2008, the disclosure of which are hereby incorporated by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     High resolution microscopy is used in research and development, quality assurance and production in diverse fields such as material science, life science, the semiconductor industry and the food industry. 
     Optical microscopy, dating back to the seventeenth century, has reached a brick wall defined by the wavelength of deep Ultra Violet photons, giving a finest resolution of about 80 nm. The popularity of optical microscopy stems from its relative low price, ease of use and the variety of imaging environmental parameters—all translated to availability. 
     Scanning electron microscopy provides a much finer resolution (down to a few nanometers), but in order to achieve this high resolution the inspected object must be placed in a vacuum environment. 
     Determining a Working Distance 
     In non-vacuum Scanning Electron Microscopes, the distance between the object and the microscope (also referred to as “working distance”) is of the order of a few to tens microns. Knowing the exact working distance is important for resolution and contrast optimization, for safe imaging of an object without contacting the optics of the microscope, for reducing contamination generated by such a contact, and for generating a focused image by setting the focusing lens accordingly. There is a growing need to provide a fast and accurate method and system for determining the working distance. 
     Reducing the Working Distance 
     The working distance between the object and the optics of the microscope should be as small as possible but large enough to prevent the object from contacting the microscope or otherwise contaminating the microscope. There is a growing need to provide an optimal trade off between the working distance and contamination hazards. 
     SUMMARY OF THE INVENTION 
     According to an embodiment of the invention a method is provided. The method is for observing an object that is positioned in a non-vacuum environment, the method includes: generating an electron beam in the vacuum environment; scanning a region of the object with the electron beam while the object is located below an object holder; wherein the scanning comprises allowing the electron beam to pass through an aperture of an aperture array, pass through an ultra thin membrane that seals the aperture, and pass through the object holder; wherein the ultra thin membrane withstands a pressure difference between the vacuum environment and the non-vacuum environment; and detecting particles generated in response to an interaction between the electron beam and the object. 
     According to an embodiment of the invention a scanning electron microscope is provided. The scanning electron microscope includes: an electron beam source positioned in a vacuum environment; the electron beam source is adapted to generate an electron beam; an interface between the vacuum environment and a non-vacuum environment in which an object is positioned, the interface comprises an aperture array sealed by an ultra thin membrane that is substantially transparent to the electron beam and withstands a pressure difference between the vacuum environment and the non-vacuum environment; an object holder; a scanner that scans a region of the object with the electron beam while the object is located below the object holder; wherein the electron beam passes through an aperture of the aperture array, passes through the ultra thin membrane, and passes through the object holder; and a detector that detects particles generated in response to an interaction between the electron beam and the object. 
     According to an embodiment of the invention a method is provided. The method is for observing an object that is positioned in a non-vacuum environment, the method includes: illuminating an area of an object with an electron beam; wherein the electron beam is generated in the vacuum environment and passes through an aperture of an aperture array and passes through an ultra thin membrane that seals the aperture; wherein the ultra thin membrane withstands a pressure difference between the vacuum environment and the non-vacuum environment; detecting particles generated in response to an interaction between the electron beam and the object; and determining a distance between the object and the ultra thin membrane in response to detected particles. 
     According to an embodiment of the invention a scanning electron microscope is provided. The scanning electron microscope includes: an electron beam source positioned in a vacuum environment; the electron beam source is adapted to generate an electron beam; optics configured to direct the electron beam towards an area of the object; an interface between the vacuum environment and a non-vacuum environment in which an object is positioned, the interface comprises an aperture array sealed by an ultra thin membrane that is substantially transparent to the electron beam and withstands a pressure difference between the vacuum environment and the non-vacuum environment; at least one detector that detects particles generated in response to an interaction between the electron beam and the object; and a controller configured to determine a distance between the object and the ultra thin membrane in response to detection signals generated by the at least one detector. 
     According to an embodiment of the invention a method is provided. The method is for aligning an electron beam and an aperture of an aperture array, the method includes: obtaining an image of a first area of the aperture array; wherein the first area comprises multiple apertures of the aperture array; calculating a spatial relationship between a selected aperture of the multiple apertures and a reference location within the first area; aligning an electron beam with the reference location in response to the spatial relationship; and obtaining an image of a region of an object that is positioned in a non-vacuum environment; wherein the obtaining comprises scanning the region by an electron beam that is generated in a vacuum environment, passes through the selected aperture and passes through an ultra thin membrane that seals the selected aperture; wherein the ultra thin membrane withstands a pressure difference between the vacuum environment and the non-vacuum environment. 
     According to an embodiment of the invention a scanning electron microscope is provided. The scanning electron microscope includes: an electron beam source positioned in a vacuum environment; the electron beam source is adapted to generate an electron beam; an interface between the vacuum environment and a non-vacuum environment in which an object is positioned, the interface comprises an aperture array sealed by an ultra thin membrane that is substantially transparent to the electron beam and withstands a pressure difference between the vacuum environment and the non-vacuum environment; optics configured to scan, with an electron beam, a first area of the aperture array and scan a second area of the aperture array; wherein the first area comprises multiple apertures of the aperture array; wherein the second area is smaller than the first area and comprises a selected aperture; at least one detector that detects particles generated in response to an interaction between the electron beam and at least one entity out of the interface and the object; and a controller configured to: calculate a spatial relationship between a selected aperture of the multiple apertures and a reference location within the first area; and control an alignment of the electron beam with the reference location in response to the spatial relationship. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  illustrates a portion of a scanning electron microscope according to an embodiment of the invention; 
         FIG. 2  illustrates a portion of a scanning electron microscope according to another embodiment of the invention; 
         FIG. 3  illustrates an object, a sample holder, non-vacuum environment and a chamber according to an embodiment of the invention. 
         FIG. 4  illustrates a chamber that includes a force applying component according to an embodiment of the invention; 
         FIG. 5  is a cross section of an object that is an electrochemical cell and of a sample holder according to an embodiment of the invention; 
         FIG. 6  illustrates a method for observing an object that is positioned in a non-vacuum environment, according to an embodiment of the invention; 
         FIGS. 7 and 8  illustrate a first area of an aperture array according to embodiments of the invention; 
         FIG. 9  illustrates a method for aligning an electron beam and an aperture of an aperture array, according to an embodiment of the invention; 
         FIG. 10  illustrates an object holder and multiple objects according to an embodiment of the invention; 
         FIG. 11  illustrates a portion of a scanning electron microscope and various particles according to an embodiment of the invention; 
         FIG. 12  illustrates a relationship between working distance and electron distribution according to an embodiment of the invention; 
         FIG. 13  illustrates a ratio between the fraction of electrons that do not pass through an aperture array and the fraction of electrons that pass through the aperture array, according to an embodiment of the invention; 
         FIG. 14  illustrates a contrast, according to an embodiment of the invention; 
         FIG. 15  illustrates a method for observing an object that is positioned in a non-vacuum environment, according to an embodiment of the invention; 
         FIG. 16  illustrates an effect of placing a shutter on the collection efficiency according to an embodiment of the invention; 
         FIG. 17  illustrates a relationship between working distances and a calculated shutter diameter for onset of signal saturation (equilibrium point) according to an embodiment of the invention; 
         FIG. 18  illustrates a detector that includes two concentric annular detector elements (electrodes) according to an embodiment of the invention; 
         FIG. 19  illustrates collection efficiencies for the detector of  FIG. 18 , as a function of working distance according to an embodiment of the invention; 
         FIG. 20  illustrates an off axis illumination according to an embodiment of the invention; 
         FIG. 21  illustrates an annular BSE detector that has two segments according to an embodiment of the invention; 
         FIG. 22  illustrates an off-axis illumination according to an embodiment of the invention; 
         FIG. 23  illustrates a relationship between a difference signal and working distance for off axis illumination according to an embodiment of the invention; 
         FIG. 24  illustrates on-axis and off-axis illumination according to an embodiment of the invention; and 
         FIG. 25  illustrates a scanning electron microscope and another microscope according to an embodiment of the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE DRAWINGS 
     According to various embodiments of the invention a scanning electron microscope and a method are provided. An object is placed, in a non-vacuumed environment, underneath an object holder that may include at least one partially transparent foil. The foil can be of the types commonly used in transmission electron microscope (TEM) grid, with the object placed onto the side far from the microscope. (It is so-called “inverted”). Alternatively, the object can be placed on top of foil that is then flipped, turned or otherwise inverted. 
     The object holder may include at least one fully transparent portion, at least one partially transparent portion or a combination thereof. 
     This configuration allows placing non-solid objects such as gels, liquids, biological cells to be placed such as to contact a lower side of the sample holder and this sample holder can be positioned at small distance from the scanning electron microscope optics (from the interface between a vacuum environment and a non-vacuum environment) thus minimizing the possibility of contact with the scanning electron microscope. This configuration also allows placing objects such as powders, flakes, or particular small crystals. 
     This so called inverted configuration prevents contamination of the scanning electron microscope optics while providing a small working distance. 
     The inverted configuration also facilitates having the sample in a separate environment (also referred to as mini-environment) and controlling a temperature of the mini environment, controlling a pressure maintained in the mini environment, controlling a gas composition of the mini environment, providing solvents to the mini environment and applying mechanical stress levels on the object or within the mini environment. The inverted configuration also facilitates the addition of chemicals to the mini environment whether in gas, liquid or solid particles form. 
     The inverted configuration also allows connecting the sample electrically to testing equipment. The electrical connectors can be connected to the lower part of the object holder or integrated within. 
     The inverted configuration also allows irradiating the sample with photons by a light source that can be a part of another microscope or inspection tool. 
     All the manipulations mentioned above in relation to the mini environment can be executed prior to imaging with the electron microscope or during imaging which allows to image changes in real time. 
     The sample holder can be connected to, or placed on, two or three axes stage (such as a XYZ stage) that can move the object holder and hence move the object for various purposes such as but not limited to alignment with a selected aperture, moving the object to be inspected by another inspection tool or microscope, and the like. 
     According to an embodiment of the invention the sample holder can be moved (while maintained in a mini-environment) to an imaging or measurement probe or tool located on a different platform or different system. 
     The object holder can be made of a very thin foil that has minimum impact on the image resolution and contrast. The foil can be transparent both for photons and electrons with typical energies of 5-30 kV. 
     The object holder is located in the non-vacuum environment and does not need to withstand a pressure gradient between the vacuum environment and the non-vacuum environment—thus simplifying its manufacturing process and expanding the range of materials that can be used. 
     The foil can also be reinforced with a grid structure which can be placed on either the side facing the electron microscope, the side opposite the electron microscope or on both sides. 
     The inverted sample holder can be made of multiple compartments enabling putting multiple samples on the same holder for analysis. 
     The inverted configuration facilitates a scanning of an object that is an electrochemical cell and generating images (in real time) of electrochemical processes for example deposition. One of the electrodes of the electromechanical cell can be shaped as a grid and can be used to support a foil of the object holder. This electrode can be imaged with the scanning electron microscope. Another electrode of the electrochemical cell may contact a solution positioned at a lower part of the electrochemical cell. 
     The inverted configuration also facilitates connecting the sample to an external electrical power source either having the entire object at a desired potential or connecting more than a single electrode for creating potential differences on the foil. 
     According to an embodiment of the invention a method is provided. The method may include placing an object such as a non-solid object on a lower side of an object holder such that the object contacts at least one partially transparent portion (such as a foil) of the object holder; imaging the object, while placed on the lower side of the sample holder by at least one scanning electron microscope. 
     Scanning Electron Microscope 
       FIG. 1  illustrates a portion of scanning electron microscope  100  according to an embodiment of the invention.  FIG. 2  illustrates a portion of scanning electron microscope  100  according to another embodiment of the invention. 
       FIG. 1  illustrates an “inverted configuration” of scanning electron microscope  100  while  FIG. 2  illustrates a non-inverted configuration of scanning electron microscope  100 . It is noted that a scanning electron microscope that can operate in both configurations as well as a scanning electron microscope that can operate at only one of these configurations can be provided without departing from the spirit of the invention. 
     The inverted configuration allows a reduction of the working distance by placing the object below an object holder—placing the object at a side that does not face the interface between the vacuum and non-vacuum environments. 
     Scanning electron microscope  100  includes electron beam source  110 , interface  120 , object holder  130 , optics  140 , scanner  140  and one or more detectors such as detectors  150  and  160 . Scanner  140  is illustrated as including multiple deflection coils  140 . It can also include a mechanical stage that can move object holder  130  and therefore object  10 . 
     Electron beam source  110  is positioned in vacuum environment  170 . Electron beam source  110  is adapted to generate electron beam  20 . 
     Interface  120  is positioned between vacuum environment  170  and non-vacuum environment  180 . Object  10  is positioned in non-vacuum environment  170 . Interface  120  includes aperture array  122  that is sealed by an ultra thin membrane  124  that is substantially transparent to electron beam  20  and withstands a pressure difference between vacuum environment  170  and non-vacuum environment  180 . 
     Object holder  130  holds object  10 .  FIG. 1  illustrates object holder  130  as placed above object  10 . 
     Scanner  140  scans a region of object  10  with electron beam  20  while object  10  is located below object holder  130 . Object  10  is below object holder  130  in a sense that it is not placed on an upper part of object holder  130 —it does not face the interface  120 . Accordingly, electron beam  20  has to pass through object holder  130  or a dedicated portion thereof in order to interact with object  10 . 
     When scanner  140  scans the region of object  10  electron beam  20  passes through one or more apertures of aperture array  122 , passes through ultra thin membrane  124 , and passes through object holder  130 . Object holder  130  can include portions that are transparent or substantially transparent to electron beam  20 . Object holder  130  can include a grid that can contact object  10 . 
     Object  10  may be a non-solid object such as fluid, gel, emulsion, biological cell and the like. Object  10  may also be one or more small particles, a powder and the like. 
     Object  10  may be placed above object holder  130  and object holder  130  can be rotated or otherwise moved or manipulated so that when being scanned by electron beam  20  it is below object holder  130 . Alternatively, object  10  may be provided to the lower part of object holder  130  without rotating or otherwise moving object holder  130 . 
     According to an embodiment of the invention object  10  does not contact the lower part of object holder  130  but contacts a gel or emulsion or other non-solid material that contacts object holder  130 . Object holder  130  can be shaped to contact a non-solid entity in which the object is inserted. It can include a rigid lower side in order to provide better connectivity to the non-solid entity. 
     One or more objects can be located below object holder at a time. Accordingly, object holder  130  can include multiple object holder regions, each region is shaped to contact an object  10 .  FIG. 10  illustrates object holder  130  as holding multiple objects  11  in multiple regions  139 . 
     Referring back to  FIG. 1 , each of detectors  150  and  160  detect particles generated in response to an interaction between electron beam  20  and object  10 . Detector  150  is located in vacuum environment  170  while detector  160  is located in non-vacuum environment  180 . 
       FIG. 1  illustrates detector  150  as having an aperture through which electron beam  20  passes. It is referred to as “in lens” detector. It can include different detector elements—each capable of generating its own detection signals. Each detector element can also be referred to as a detector. 
     Detector  160  is positioned in non-vacuum environment  180 . It can be connected to interface  120  and measure a current that flows though interface  120 . Additionally or alternatively, detector  160  can detect electrons that do not pass through aperture array  122  in other manners. 
     Scanning electron microscope  100  can be used for various purposes, including but not limited to: (i) image voltage contrast in air; (ii) electron beam lithography on photo resist in air; (iii) high resolution imaging of wafers and processes which are incompatible with vacuum such as a photo resist before curing; (iv) image and analyze wafers and processes which are impacted by the vacuum environment, or wafers and processes which are sensitive to formation of adhesion of a monolayer of contamination molecules; (v) image and analyze large specimen such as solar panels and flat panel display; (vi) image and analyze particles in a solution; (vii) image histology and pathology slides; (viii) image biological cell; (ix) excite X-ray emission for material analysis, whereas an image can be used to find a known location and generate an analysis on an exact location; (x) excite X-ray emission for thickness measurement, whereas an image can be used to find a known location and generate an analysis on an exact location; (xi) excite X-ray emission for density measurement, whereas an image can be used to find a known location and generate an analysis on an exact location; (xii) image and analyze side walls, whereas an image can be used to find a known location and generate an analysis on an exact location, (xiii) image and measure thickness of side walls, whereas an image can be used to find a known location and generate an analysis on an exact location, and the like. 
     Aperture Array  122  and Ultra Thin Membrane  124   
     The term aperture array means any arrangement (ordered or non-ordered) of apertures. The apertures of the array can be sealed by one or more ultra thin membranes. 
     Conveniently, the ultra thin membrane is thinner than a 100 nanometers and is made of low density material such as Carbon or SiN. 
     When multiple apertures are provided each aperture can be sealed by its own membrane, although this is not necessarily so. A single membrane can seal multiple apertures. According to an embodiment of the invention the membrane can be connected to a very thin grid that defines multiple apertures. 
     The ultra thin membrane seals an aperture while withstanding the pressure difference between the vacuum environment and the non-vacuum environment. 
     The ultra thin membrane is used because it has the minimal impact on the electron spot size. For better performance it is advantageous to use higher electron accelerating voltages, preferably 20 kV and higher. Another advantage of using ultra thin membrane is that the electrons used to generate the image can be efficiently collected with detectors situated in the vacuum environment. 
     The aperture array can include an aperture array of different sized apertures that are sealed by membranes of different areas and thickness. The different apertures can be positioned at the same plane, but this is not necessarily so. For example, apertures and membranes can be positioned in a staggered manner. The apertures positioned at the center of the aperture array can be lower than aperture positioned near the edges of the aperture array. The apertures of the array can be positioned in different planes that are arranged in a symmetrical manner about the center of the aperture array or in relation to a symmetry axis, but this is not necessarily so. 
     Different apertures can be sealed by ultra thin membranes (or different portions of the same ultra thin membrane) of the same thickness. Alternatively, different apertures can be sealed by ultra thin membranes (or different portions of the same ultra thin membrane) of different thickness. 
     The thickness can be responsive to the size of the aperture. Larger apertures may be sealed by thicker membranes. Larger apertures provide larger field of view but thicker membranes reduce the resolution. Accordingly, a system that includes membranes of different thickness and apertures of different areas can provide multiple trade-offs between field of views and resolution. 
     The different apertures can be accessed by moving the apertures to the electron beam position, and/or by deflecting the electron beam. According to another embodiment of the invention an aperture array includes evenly sized apertures and conveniently evenly sized membranes. 
     The apertures of the aperture array can all have the same shape but this is not necessarily so. Non limiting examples of apertures shapes include circles, ellipse, square, triangle, rectangle, and the like. 
     An entire area of the object can be imaged by scanning that area by an aperture array. The scanning axis can be parallel a longitudinal axis or a latitudinal axis of the aperture array but can also be oriented in relation to these arrays. 
     It is noted that when virtually combining the field of view provided by each of the apertures a relatively large (though not continuous) field of view can be obtained. A non-continuous image of sub-areas of the object can be used during navigation stages. 
     An aperture array can be manufactured in various manners including deposition and etch back. The deposition includes depositing one or more ultra thin membrane on an aperture array. The etch back process includes etching a plate in order to form multiple apertures. 
     It is noted that different aperture arrays or different aperture can be used in an interchangeable manner. An interface of a scanning electron microscope can include multiple aperture arrays and at a given point of time one (or more) aperture array can be illuminated by one or more electron beams. 
     Scanner  140   
     Scanner  140  can scan an area or a region of object  10  by at least one of the following or a combination of both: (i) electrostatic scanning of electron beam  20 ; (ii) mechanical scanning of the object with electron beam  20  in spot mode to form an image, which can be useful if one utilizes a very small aperture and wants to generate an image with field of view larger than the size of the aperture; (iii) mechanical scanning of the microscope with electron beam  20  in spot mode to form an image which can be useful if one utilizes a very small aperture and wants to generate an image with field of view larger than the size of the aperture; (iv) mechanical scanning of the aperture or window simultaneously with electrostatic scanning of electron beam  20  so that electron beam  20  follows the window position; (v) scanning the aperture by magnetic scanning. 
       FIG. 1  illustrates scanner  140  as placed between interface  120  and electron beam source  110  but this is not necessarily so. For example—scanner  140  can include a stage (such as stage  240  of  FIG. 2 ) that can move object  10 . The stage can be connected to object holder  130  or be a part of object holder  130 . 
     Detectors  150 ,  160   
     Particles such as electrons that interact with the object can cause various particles to be scattered or reflected from the object. The interaction can generate secondary electrons, backscattered electrons, characteristic X-rays and in some cases Cathodoluminescence. The Cathodoluminescence can be either a surface property or caused due to light emission from markers or labeling molecules. The emitted signal is detected with the aid of one of the mentioned above detectors. 
       FIG. 1  illustrates scanning electron microscope  100  as including detector  150  and detector  160 . It is noted that scanning electron microscope  100  can include more or less detectors. For example—scanning electron microscope can have more than a single detector within vacuum environment  170  and, additionally or alternatively, have more than a single detector in non-vacuum environment  180 . 
     According to embodiments of the invention, scanning electron microscope  100  can have only vacuum environment detectors or have only non-vacuum environment detectors. A combination of both can also be provided thus one or more detector is positioned in the non-vacuum environment while one or more other detectors are positioned in the vacuum environment. 
     Locating one or more detector such as vacuum environment detector  150  in vacuum environment  170  can facilitate small and even very small working distances between the object and one or more apertures, thus contribute to the resolution of the image. Placing detectors in the vacuum environment also enables to use detectors that are less compatible with air such as using coatings which easily oxidize. 
     It is noted that using different detectors can provide more information about the illuminated area of the object and that multiple detectors can be activated simultaneously. 
     Detectors  150  and  160  can detect the same type of particles. According to another embodiment different detectors of scanning electron microscopes can detect different types of particles. 
     For example—one detector of scanning electron microscope  100  can detect backscattered electrons (BSE). A BSE detector can be located between ultra thin membrane  124  and an objective lens (not shown) of scanning electron microscope  100 . The BSE detector can have an annular shape that defines an opening enabling electron beam  20  to pass. The BSE detector can also be segmented to enhance topography information. 
     According to another embodiment one detector can detect electrons and another detector detects light. Both detectors may operate simultaneously. 
     A parabolic mirror (not shown) located between ultra thin membrane  124  and an objective lens of scanning electron microscope  100 , having an opening enabling electron beam  20  to pass will collect the light to a photomultiplier placed to the side of the electron path. 
     Scanning electron microscope  100  can include one or more X-ray detectors that can assist in material analysis. Integrating such analysis to an imaging engine permits localization of the object to be analyzed enabling higher sensitivity for smaller objects as opposed to macroscopic analysis. Another possibility is to use emitted X-rays for image generation which is commonly referred to as X-ray mapping. 
     For analysis with low resolution where working distance of &gt;100 microns can be applied, an X-ray detector can be located outside the vacuum environment. It would be preferable to use an annular detector to increase the collection efficiency. 
     For analysis with high resolution, where smaller working distance has to be applied, lets say &lt;100 microns, the detector will be situated inside the vacuum. If a side detector is used it can be in a configuration where backscattered electrons, secondary electrons and light can be detected. An alternative arrangement where emphasis is on high X-ray collection efficiency is to use an annular X-ray detector such as a multi cell Silicon drift detector (SDD) manufactured by PN Sensor, Germany. 
     Vacuum Environment  170   
     A scanning electron microscope column can operate under vacuum. The column of scanning electron microscope  100  can include multiple differentially pumped spaces that are separated by a non-sealed aperture. It is noted that the sealing provided by interface  120  can render such a partition unnecessary. It is noted that the vacuum of vacuum environment  170  can be provided by one or more pumps such as an ion pump, a turbo pump and the like. Since the system is isolated a microscope can be designed without a pump as done in a CRT. 
     Non-Vacuum Environment  180   
     The environment between the sample and the column can be of any composition. In particular, it can be filled with air, at least partially filled with nitrogen or dry nitrogen where the efficiency of light emission due to secondary gas excitation is high because the oxygen which is a quencher of this process is absent. Non-vacuum environment  180  can be at least partially filled with inert gases and in particular He or mixture of He where the mean free path of the electron is higher improving the signal to noise at larger working distances. 
     Inverted Configuration 
       FIG. 3  illustrates object  10 , sample holder  130 , non-vacuum environment  180  and chamber  210  according to an embodiment of the invention. 
     Chamber (also referred to as micro-chamber)  210  can define a space in which mini environment  220  can exist. Mini environment  220  is defined around object  10 . Mini environment  220  is smaller than non-vacuum environment  180  and may be even much smaller. For example it can be 5%, 10%, 20% or 30% of the non-vacuum environment, but this is not necessarily so. 
     Chamber  210  can control one or more characteristics of mini environment  220  by introducing one or more chemicals, by heating or cooling mini environment  220 , by drying of introducing vapors such as to determine a humidity of mini environment  220 , by directing radiation towards object  10 , by applying force (for example squeezing object  10  against object holder  130 ), and the like. 
     Chamber  210  includes frame  218 , inlet  212 , outlet  214 , temperature affecting element  216  (such as a cooling element, a heating element or both). Inlet  212  can be used to inject one or more chemicals, gases, fluids and the like. 
       FIG. 3  illustrates sample holder  130  as including a transparent portion  132  (such as a semi transparent film) and sample holder base  134 . Object  10  is positioned below transparent portion  132 . 
       FIG. 4  illustrates chamber  210  that includes force applying component  230  according to an embodiment of the invention. Force applying component  230  can apply force on object  10  while object  10  is within mini environment  220 . It is noted that scanning electron microscope  100  can include a force applying component even without having chamber  210 . 
       FIG. 5  is a cross section of an object  10  that is an electrochemical cell  250  and of sample holder  130  according to an embodiment of the invention. 
     Electromechanical cell  250  includes electrolyte solution  251  that contacts semi transparent film  136  of sample holder  130  that in turn is supported by conducting grid  138 . Conducting grid  138  serves as one of the electrodes of electromechanical cell  250  and can be electrically coupled (via connector  252 ) to an electrical device (not shown). Second electrode  253  of electrochemical cell  250  can contact electrolyte solution  251 . Sample holder base  134  can be made of an insulating material. 
       FIG. 6  illustrates method  600  for observing an object that is positioned in a non-vacuum environment, according to an embodiment of the invention. 
     Method  600  may start by initializing stage  610 . Stage  610  can include placing the object below the object holder, affecting a parameter of a mini environment in which the object is positioned and the like: 
     Stage  610  can include placing the object such as to contact a lower side of the object holder, placing the object such as to contact a foil of the object holder, placing the object on the object holder and than rotating or otherwise manipulating the object holder so that the object is placed below the object holder. 
     Stage  610  can include placing the object within a non-solid entity that contacts a lower side of the object holder and scanning a region of the object. 
     Stage  610  may include placing multiple objects below the object holder. 
     Stage  610  may include imaging the object by a low magnification technique such as optical microscope to facilitate navigation to the object. Alternatively, stage  610  can include navigating to the object in response to an image of the object (or object vicinity) that may be acquired by applying an imaging process that differs (at least by its resolution) from the imaging process that is executed during stages  630 - 650 . 
     Stage  610  is followed by stage  620  of generating an electron beam in the vacuum environment. Stage  620  can include generating multiple electron beams. 
     Stage  620  is followed by stage  630  of scanning a region of the object with the electron beam while the object is located below an object holder. Stage  630  of scanning includes allowing the electron beam to pass through an aperture of an aperture array, pass through an ultra thin membrane that seals the aperture, and pass through the object holder. The ultra thin membrane withstands a pressure difference between the vacuum environment and the non-vacuum environment. 
     Stage  630  can include scanning one or more regions of one or more objects by one or more electron beams. 
     Stage  630  may include at least one of the following or a combination thereof: (i) scanning at least one region of the object by deflecting the at least one electron beam and introducing a corresponding mechanical movement of the aperture array; (ii) scanning at least one region of the object by deflecting the at least one electron beam and introducing a corresponding mechanical movement of the aperture array; wherein a component that includes the aperture array is flexibly connected to another component of an interface that separates the vacuum environment from the non-vacuum environment; (iii) scanning multiple regions of the object by deflecting  15 —the at least one electron beam that pass through multiple apertures of the aperture array; (iv) scanning multiple regions of the object by deflecting the at least one electron beam that pass through multiple apertures of the aperture array and introducing a corresponding mechanical movement of the aperture array; (v) scanning a region of the object by deflecting the at least one electron beam by a deflector positioned within the vacuum environment. 
     Stage  630  is followed by stage  640  of detecting particles generated in response to an interaction between the electron beam and the object. 
     Stage  640  is followed by stage  650  of processing detection signals generated as response to the detection of particles. Stage  650  can include generating an image of the region of the object, determining a state of the object, or evaluating other parameters of the object. The processing can be preceded by, followed by or executed in parallel of storing the detection signals. 
     According to an embodiment of the invention method  600  includes stage  670  of imaging the object by another microscope while the object is located below the object holder. The other microscope can be an optical tool that directs light towards the lower part of the object.  FIG. 25  illustrates another microscope such as optical microscope  2400  that images object  10  while object is located below object holder  130 . Optical microscope  2400  directs a light beam  2410  that propagates along an optical axis that is normal to object  10 —as illustrated by dashed line  2410 .  FIG. 25  illustrates optical microscope  2400  and scanning electron microscope  100  as having optical axes that are opposite to each other. It is noted that the optical axis of these microscopes can be oriented in relation to each other by angles that may differ from 180 degrees. Both microscopes  100  and  2400  can image or scan object simultaneously, in a partially overlapping manner or in a non-overlapping manner in which each microscope images or scans object  10  during non-overlapping periods. 
     Stage  610  can include placing the object within a chamber that defined a mini environment that at least partially surrounds the object. Stage  600  can include stage  680  of controlling at least one characteristic of the mini environment during the scanning of the region of the object. The controlling can include inducing at least one chemical within the mini environment, applying force on the object while the object is scanned, changing a temperature of the mini environment, changing a humidity of the mini environment, and the like. 
     Alignment of Electron Beam  20   
     Referring back to  FIG. 1 , each of detectors  150  and  160  can send detection signals that may be processed by controller  190  in order to generate an image of the area of object  10 . 
     An area of the object can be scanned by directing electron beam  20  through a selected aperture of aperture array  122 . It can be beneficial to align electron beam  20  with the selected aperture (denoted  123 ). 
       FIGS. 7 and 8  illustrate first area  121  of aperture array  122  according to embodiments of the invention. First area  121  includes apertures  1221 ,  1222 ,  123  and  1224 .  FIG. 8  illustrates first area  121  as further including alignment target  125 . 
     The alignment process can start by scanning with electron beam  20  first area  121  of aperture array  122 . First area  121  includes multiple apertures of aperture array  122 . First area defines a first field of view of scanning electron microscope. A second field of view of scanning microscope can be defined when the object  10  is scanned through a selected aperture  123 . 
     A detector, such as detector  150  or  160 , detects particles generated in response to an interaction between electron beam  20  and first area  121  and sends detection signals to controller  190 . Controller  190  may process the detection signals to provide an image of first area  121  and to calculate a spatial relationship (distance, relative angle) between selected aperture  123  and a reference location within first area  121 . The reference location can be an alignment target  125 , a corner of first area  121 , an aperture that has a unique shape and the like. 
     Once the spatial distance is calculated controller  190  can control a completion of the alignment process. The controller  190  can send control signals to deflectors, to a mechanical unit that can introduce a mechanical movement between interface  120  and scanner  140 , and the like. Once the alignment process ends the scanning electron microscope  100  can obtain an image of an area of object  10 . The area can include the entire object  10 , a small portion of the object or a large area of object  10 . The area of object  10  is scanned by scanning a second area  127  of aperture array  122 . Second area  127  is smaller than first area  121  and can correspond to selected aperture  123 . 
     The alignment can be done by scanner  140  but can also be done by using mechanical movements. The alignment can utilize at least one of the following: (i) alignment coils ensuring that the one or more electron beams are aligned with the one or more apertures; (ii) mechanical movement of the aperture array (or at least the one or more relevant apertures); and (iii) mechanical movement of the objective or a permanent magnet relative to the aperture array. The advantage is that the moving part is not part of the vacuum sealing. 
     The mechanical movement of aperture array  122  (or relevant apertures) can be achieved by using a flexible connector between the aperture array and another part of the interface. The flexible connector can be moved by a motor (piezo-motor, linear motor and the like). 
     Either one of the alignment methods mentioned above or combination of one or more alignment methods can be used also to select one or more apertures of an aperture array. 
     In case of mechanical alignment of the aperture, the movement can be motorized, controlled by a controller and positions can be calibrated or preset to allow seamless work. 
     A flexible connector can be also used to connect aperture array  122  to frame  126  of  FIG. 1 . The flexible connector can control the distance between the object and the aperture by moving the aperture array  122  in the direction perpendicular to the plane of object  10 . This has the advantage of maintaining a fixed distance between the object and the aperture while moving a small mass. 
       FIG. 9  illustrates method  900  for aligning an electron beam and an aperture of an aperture array, according to an embodiment of the invention. 
     Method  900  starts by stage  910  of obtaining an image of a first area of the aperture array; wherein the first area includes multiple apertures of the aperture array. The first area can include the entire aperture array or a portion thereof. The first are can be selected such as to include a selected aperture and at least one other aperture or reference location. 
     Stage  910  is followed by stage  920  of calculating a spatial relationship between a selected aperture of the multiple apertures and a reference location within the first area. The reference location can be an alignment target or any other unique point within first area. 
     Stage  920  is followed by stage  930  of aligning an electron beam with the reference location in response to the spatial relationship. 
     At the end of stages  920 - 930  an alignment is achieved. Once such an alignment is achieved method  900  can proceed to obtaining an image of a region of an object. Stage  930  can include applying any of the mentioned above alignment techniques. 
     Stage  930  is followed by stage  940  of obtaining an image of a region of an object that is positioned in a non-vacuum environment. Stage  940  includes scanning the region by an electron beam that is generated in a vacuum environment, passes through the selected aperture and passes through an ultra thin membrane that seals the selected aperture. The ultra thin membrane withstands a pressure difference between the vacuum environment and the non-vacuum environment. 
     Stage  940  may includes scanning a second area of the aperture array. The second area is smaller than the first area. 
     Working Distance 
     Referring back to  FIG. 1 , scanning electron microscope  100  can determine the working distance (defined between object  10  and interface  120  or any part thereof) by evaluating the relationship between two or three elements out of: (i) an amount of electrons generated by an interaction between an object and the electron beam; (ii) an amount of electrons that pass through the aperture array; (iii) an amount of electrons that do not pass the aperture array but hit the frame that supports the aperture array. 
     It is noted that these amount can be measured or estimated in various manners. For example, the amount of electrons that pass through aperture array  122  can be estimated by measuring an amount of electrons that are detected by one or more detectors (such as detector  150 ) located in vacuumed environment  170 . Yet for another example, the amount of electrons that do not pass through aperture array  122  can be estimated by measuring an amount of electrons that impinge on aperture array  122  or on frame  126  that at least partially surrounds aperture array  122 . The overall amount of electrons can be estimated by measuring the intensity of electron beam  20  and by estimating the yield of object  10 —the yield being the percentage of electrons that are scattered or reflected from object  10 . This evaluation can involve illuminating a target of known characteristics (for example—of known yield) and measuring electrons that are emitted from the target, electrons scattered from the target or current that flows through the target. 
     Each relationship between the mentioned above amounts of electrons reflects the angular distribution of electrons and this angular distribution is responsive to the working distance. Each working distance may be characterized by a unique relationship between the mentioned above amounts of electrons. 
     Controller  190  may determine the distance by at least one of the following manners or combination thereof: (i) in response to a comparison between detection signals generated when the electron beam is positioned in different locations in relation to the aperture; (ii) in response to a comparison between detection signals generated when the electron beam illuminates the object at different illumination paths; (iii) in response to a comparison between shadows that appear in different locations of an image of the area of the object; (iv) by finding a saturation point; and (v) in response to a size of the aperture and in response to a saturation point; wherein the size of the aperture is set by a shutter. 
     Another to various embodiments of the invention the working distance can be determined in response to a change of the collection angle for different parts of the window.  FIG. 24  illustrates electron beam  20  that is positioned at the center of aperture  122  and electron beam  20 ′ that is positioned near the right side of aperture  122 . Electron beam  20  causes particles  445  to impinge on a first detection portion  151  and particles  444  to impinge on second detector portion  152 . Electron beam  20 ′ causes particles  455  to impinge on a first detection portion  151  and particles  454  to impinge on second detector portion  152 . 
     The available collection angle of particles associated with electron beam  20  is larger that the available collection angle associated with electron beam  20 ′. This difference is larger for longer working distances and can be manifested in shading of the image near the edges of aperture  122 . The dependence of the width of the shading as a function of working distance for a given window geometry can be found either by simulation or calibration. For the measurement itself one can use either discrete points, a line scan crossing the entire window or the entire image. 
       FIG. 11  illustrates a portion of scanning electron microscope  100  and various particles according to an embodiment of the invention. 
       FIG. 11  illustrates detector  160  as a current meter that measures the current that flows through frame  126  that is connected to aperture array  126 .  FIG. 11  also illustrates electrons that pass through aperture array  122  and are detected by detector  150 .  FIG. 126  also illustrates a collection angle  1111 . 
     If, for example, the aperture array includes a single circular aperture that has a radius of r, and the working distance is d then the electrons that will pass through this aperture are within collection angle  1111  (defined in relation to a propagation axis of electron beam  20 ) that equals arctangent(r/d). Electrons that are outside this angle will hit frame  126 . The collection angle is also referred to as solid angle. 
     If the working distance is large enough then the relationship between electrons that pass aperture array  122  and those who do not pass it are dependent upon the collection angle and are not substantially affected by the shape of frame  126 . On the other hand, at relatively small working distances the shape of frame  126  and especially a slope (if such exists) of the inner edges of frame  126  may affect the relationship between the amount of electrons that pass through aperture array  122  and those who does not pass it. 
     The distribution of particles emitted as a result of an interaction with object  10  can be known in advance and this distribution can assist, once the relationship between the amounts of electrons is measured, to determine the working distance. For example, back scattered electrons are emitted from object  10  with a Lambert (cos θ) distribution, wherein θ is the angle between the emitted electron and the normal to object  10 . The calculation of working distance d can also be responsive to known losses. 
     Table 1 illustrates a relationship between working distances (expresses as multiplication of radius r of a circular aperture), collection angle, percent of electron that pass through the aperture array, percent of electron that do not pass through the aperture array, ratio of electrons that pass aperture array and those who do not pass through the aperture array, and contrast. The contrast is the ratio between the difference and the average of two measurements taken at two working distances separated by r/2. 
     In a non-limiting example, if the aperture has a radius of 10 microns, the working distances span is 5 to 100 microns 
     
       
         
           
               
               
               
               
               
               
             
               
                   
               
               
                   
                   
                 %  
                 % of 
                 Ratio of 
                   
               
               
                   
                   
                 electrons 
                 electrons 
                 electrons that 
                   
               
               
                 working 
                   
                 that pass 
                 that do not 
                 pass aperture 
                   
               
               
                 distance 
                 collection 
                 through 
                 pass 
                 array and those 
                   
               
               
                 (window 
                 angle 
                 aperture 
                 aperture 
                 who do not 
                   
               
               
                 radius) 
                 (radians) 
                 array 
                 array 
                 pass 
                 contrast 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                  0.5 
                 1.107 
                 0.894 
                 0.106 
                 8.472 
                   
               
               
                  1 
                 0.785 
                 0.707 
                 0.293 
                 2.414 
                 1.11 
               
               
                  1.5 
                 0.588 
                 0.555 
                 0.445 
                 1.246 
                 0.64 
               
               
                  2 
                 0.464 
                 0.447 
                 0.553 
                 0.809 
                 0.43 
               
               
                  2.5 
                 0.381 
                 0.371 
                 0.629 
                 0.591 
                 0.31 
               
               
                  3 
                 0.322 
                 0.316 
                 0.684 
                 0.462 
                 0.24 
               
               
                  3.5 
                 0.278 
                 0.275 
                 0.725 
                 0.379 
                 0.20 
               
               
                  4 
                 0.245 
                 0.243 
                 0.757 
                 0.320 
                 0.17 
               
               
                  4.5 
                 0.219 
                 0.217 
                 0.783 
                 0.277 
                 0.14 
               
               
                  5 
                 0.197 
                 0.196 
                 0.804 
                 0.244 
                 0.13 
               
               
                  5.5 
                 0.180 
                 0.179 
                 0.821 
                 0.218 
                 0.11 
               
               
                  6 
                 0.165 
                 0.164 
                 0.836 
                 0.197 
                 0.10 
               
               
                  6.5 
                 0.153 
                 0.152 
                 0.848 
                 0.179 
                 0.09 
               
               
                  7 
                 0.142 
                 0.141 
                 0.859 
                 0.165 
                 0.08 
               
               
                  7.5 
                 0.133 
                 0.132 
                 0.868 
                 0.152 
                 0.08 
               
               
                  8 
                 0.124 
                 0.124 
                 0.876 
                 0.142 
                 0.07 
               
               
                  8.5 
                 0.117 
                 0.117 
                 0.883 
                 0.132 
                 0.07 
               
               
                  9 
                 0.111 
                 0.110 
                 0.890 
                 0.124 
                 0.06 
               
               
                  9.5 
                 0.105 
                 0.105 
                 0.895 
                 0.117 
                 0.06 
               
               
                 10 
                 0.100 
                 0.100 
                 0.900 
                 0.110 
                 0.06 
               
               
                   
               
            
           
         
       
     
       FIG. 12  illustrates a relationship between the working distance and electron distribution according to an embodiment of the invention. Curve  1210  illustrates the relative amount (fraction) of electrons that do not pass through aperture array  122  while curve  1220  illustrates the relative amount (fraction) of electrons that pass through aperture array  122 . 
       FIG. 13  illustrates a ratio between the fraction of electrons that do not pass through aperture array and the fraction of electrons that pass through aperture array  122 , according to an embodiment of the invention.  FIG. 13  includes curve  1300  that illustrated this ratio. 
       FIG. 14  illustrates a contrast; according to an embodiment of the invention.  FIG. 14  includes curve  1400  that illustrated the contrast. 
       FIG. 15  illustrates method  1500  for observing an object that is positioned in a non-vacuum environment, according to an embodiment of the invention. 
     Method  1500  starts by stage  1510  of illuminating an area of an object with an electron beam. The electron beam is generated in the vacuum environment and passes through an aperture of an aperture array and passes through an ultra thin membrane that seals the aperture. The ultra thin membrane withstands a pressure difference between the vacuum environment and the non-vacuum environment. 
     Stage  1510  is followed by stage  1520  of detecting particles generated in response to an interaction between the electron beam and the object. 
     Stage  1520  can include at least one of the following stages or a combination thereof: (i) detecting electrons generated in response to the interaction between the at least one electron beam and the object; (ii) detecting photons generated in response to the interaction between the at least one electron beam and the object; (iii) detecting X-ray emission generated in response to the interaction between the at least one electron beam and the object; (iv) detecting particles by a detector positioned within the vacuum environment; (v) detecting particles by a detector positioned within the non-vacuum environment; (vi) detecting electron current generated as a result of the interaction; and (vii) detecting Cathodoluminescence of the object, fluorescence markers or light emitted due to electron excitation of gas molecules. 
     Stage  1520  is followed by stage  1530  of determining a distance between the object and the ultra thin membrane in response to detected particles. It is noted that the distance can be determined by an approach curve generated by several measurements at different working distances or by detection signals obtained while the distance between the scanning electron microscope and the object remains the same. 
     Stage  1530  is followed by stage  1540  of responding to the determination of the working distance. Stage  1540  can include adjusting a focus of the scanning electron microscope to comply with the working distance, changing the working distance in order to achieve a desired working distance, or perform another iteration of stages  1510 - 1530 . The additional iteration can be performed in order to increase the reliability of the determination of the working distance. 
     Stage  1540  can include changing the working distance and jumping to stage  1510 . Thus additional iteration of the measurement process can be performed. This can assist in finding a saturation point or in determining the working distance-especially if only a single detector detects particles during stage  1520 . 
     Accordingly, stage  1540  can include altering the working distance and jumping to stage  1510  until a relationship between particles that pass through the aperture array and particles that not pass through the aperture array maintains constant despite the altering of the distance. This is illustrated by stage  1541  of checking if a saturation point has been detected. 
     The additional iteration can be executed after changing the working distance but this is not necessarily so. The additional iteration can assist in determining the working distance 
     Stage  1510  can include stage  1511  of illuminating an area of the object by directing the electron beam to pass through the aperture at a point that differs, from a center of the aperture. In this case stage  1520  can include stage  1521  of detecting particles by detectors that have different collection regions and stage  1530  can include stage  1531  of determining the distance in response to a relationship between detection signals generated by the detectors. 
     Stage  1530  can include stage  1532  of determining the distance in response to a difference between detection signals generated by the detectors. 
     Stage  1520  can include stage  1523  of detecting particles by at least one detector located in the non-vacuum environment and by at least one detector located in the vacuum environment and stage  1530  can include stage  1533  of determining the distance in response to a relationship between detection signals generated by the detectors. 
     Stage  1510  can include stage  1514  of illuminating the area of the object with an electron beam of a first intensity and imaging a region of the object by scanning the region with an electron beam of a second intensity; wherein the second intensity is lower than the first intensity. 
     Stage  1530  can include stage  1535  of determining the distance in response to a ratio between at least two out of: (i) amount of particles that passes through the aperture array as a result of the interaction; (ii) amount of particles that did not pass through the aperture array; and (iii) amount of particles generated as result of the interaction. 
     Stage  1520  can include stage  1526  of measuring a current that flows through at least one detector. 
     Method  1500  may include at least one of the following stages or a combination thereof: (i) comprising determining the distance in response to a comparison between detection signals generated when the electron beam is positioned in different locations in relation to the aperture; (ii) determining the distance in response to a comparison between detection signals generated when the electron beam illuminates the object at different illumination paths (different angles of incidence, different location in relation to the aperture); (iii) determining the distance in response to a comparison between shadows appearing in different locations of an image of the area of the object; (iv) finding a saturation point; (v) setting a size of the aperture by a shutter and finding a saturation point. 
       FIG. 16  illustrates an effect of placing a shutter on the collection efficiency according to an embodiment of the invention. 
     In general, the shutter defines the effective size (and even shape) of an aperture. An aperture of different effective sizes is characterized by different saturation points as illustrated by  FIG. 16 . According to an embodiment of the invention the working distance can be determined by finding the saturation point. The working distance that corresponds to a saturation point can be set by changing the effective size of the aperture—for example, by closing or opening the shutter. 
     It is assumed that a detector is placed at a distance of 4 mm above a circular aperture, the shutter is located 2 mm above the aperture and the diameter of the aperture is 250 μm. The collection efficiency dependence on the shutter opening was calculated for three different working distances—0 μm (graph  1610 ), 200 μm (graph  1620 ) and 400 μm (graph  1630 ). It is noted that for different working distances, the saturation point occurs at different shutter diameters. 
       FIG. 17  illustrates a relationship between working distances and a calculated shutter diameter for onset of signal saturation (equilibrium point) according to an embodiment of the invention. The relationship is illustrated by curve  1710 . 
       FIG. 18  illustrates a detector  1800  that includes two concentric annular detector elements (electrodes)  1810  and  1820  according to an embodiment of the invention. The inner annular electrode  1810  surrounds aperture  1830 . 
       FIG. 19  illustrates collection efficiencies for the detector of  FIG. 18 , as a function of working distance according to an embodiment of the invention. 
     Detector  1800  is located 4 mm above an aperture of diameter of 250 μm. Inner annular electrode  1810  has a diameter of 6 mm and surrounds aperture  1830  of diameter of 1 mm, and outer annular electrode  1820  has a diameter of 9 mm. 
     Curve  1910  illustrates detection signals (denoted A) generated by inner annular electrode  1810 . 
     Curve  1920  illustrates detection signals (denoted B) generated by outer annular electrode  1820 . 
     Curve  1930  illustrates the difference (denoted A-B) between detection signals generated by inner annular electrode  1810  and outer annular electrode  1820 . 
       FIG. 20  illustrates an illumination of electron beam  20  at a location that differs from the center of aperture  123  according to an embodiment of the invention. Aperture  123  is supported by frame  126  and has two axes of symmetry denoted  3  and  4 . This illumination is also referred to as off axis illumination. 
       FIG. 21  illustrates an annular BSE detector  2100  having two segments  2101  and  2102  according to an embodiment of the invention. BSE detector  2100  can be either one of detectors  150  and  160 . 
       FIG. 22  illustrates an off-axis illumination according to an embodiment of the invention. 
     Electron beam  20  is directed to a location that differs from the middle of aperture  122 . Particles such as electrons interact with object  10  and are detected by different portions (different detector elements, different electrodes)  6  and  150 . Particles that are directed towards portion  6  are denoted  444  and particles that are directed towards portion  150  are denoted  445 . 
       FIG. 23  illustrates a relationship between a difference signal and working distance for off axis illumination according to an embodiment of the invention. Curve  2310  illustrates this relationship. The difference signal grows rapidly till it reaches a maximal value at a working distance of about 150 microns. At distances above 150 microns the difference signal gradually decreases. It is noted that the working distance associated with the maximum may depend only on geometry and is not sensitive to the sample yield or to the intensity of electron beam  20 . 
     The aperture was 250 μm wide and electron beam  20  was positioned 100 microns from the center of the aperture. The difference signal is normalized by its maximal value. 
     A computer program product can be provided. It includes a computer readable medium that stores instructions for executing any of the mentioned above methods or a combination thereof. 
     However, other modifications, variations and alternatives are also possible. The specifications and drawings are, accordingly, to be regarded in an illustrative rather than in a restrictive sense. 
     The present invention can be practiced by employing conventional tools, methodology and components. Accordingly, the details of such tools, component and methodology are not set forth herein in detail. In the previous descriptions, numerous specific details are set forth, in order to provide a thorough understanding of the present invention. However, it should be recognized that the present invention might be practiced without resorting to the details specifically set forth. 
     Only exemplary embodiments of the present invention and but a few examples of its versatility are shown and described in the present disclosure. It is to be understood that the present invention is capable of use in various other combinations and environments and is capable of changes or modifications within the scope of the inventive concept as expressed herein.