Patent Application: US-93074911-A

Abstract:
new methods for phase contrast imaging in transmission electron microscopy use the imaging electron beam itself to prepare a hole - free thin film for use as an effective phase plate , in some cases eliminating the need for ex - situ fabrication of a hole and reducing requirements for the precision of the zpp hardware . the electron optical properties of the zpp hardware are modified primarily in two ways : by boring a hole using the electron beam ; and / or by modifying the electro - optical properties by charging induced by the primary beam . furthermore a method where the sample is focused by a lens downstream from the zpp hardware is disclosed . a method for transferring a back focal plane of the objective lens to a selected area aperture plane and any plane conjugated with the back focal plane of the objective lens is also provided .

Description:
a technique is provided for phase contrast imaging involving preparing a hole - free thin film for tem imaging with the electron beam itself focused on a center of the charged beam , which now does not have to be precisely aligned . the preparation involves charging the hole - free thin film , or boring a hole therethrough to produce a conventional zpp , that is uncharacteristically centered within the tem without equipment required for submicron alignment . several experiments were performed that demonstrate that a hole - free thin film in use is subject to charging that can produce a phase contrast transfer function ( ctf ) suitable for phase contrast imaging . because only the difference between the phase shift experienced by the scattered beam with respect to the incident beam results in phase contrast , the uniform films without a hole were not expected to result in a phase contrast . it is a surprising and counterintuitive result that phase contrast imaging is provided with a simple hole - free thin film . herein hole - free thin films are those that present no hole to the electron beam , even if at some peripheral edge there may be one or more holes for purposes of alignment or retention of the thin film within the tem , for example . amorphous thin films were deposited using a cryo - pumped electron beam evaporator and ultra high purity carbon source . respective thin films were formed by depositing gold and aluminum from a resistively heated source in a system with about 5 × 10 − 7 torr background pressure . all films were deposited directly on the respective selected area aperture discs to ensure good electrical contact . the thin films were held at room temperature at all times . the thin films were deposited at a high rate ( 1 to 10 å / s ) to ensure low oxidation and contamination during film deposition . the film thickness appropriate for π / 2 phase shift at 200 kv was calibrated by double - biprism electron holography in a hiatchi hf 3300 tem equipped with a cold field emission gun . in all examples herein , a jeol 2200 fs 200 kv tem / stem equipped with a schottky electron source , in - column energy filter , and a gatan ultrascan ™ 2k × 2k slow scan ccd camera , was used . a cryo polepiece was used allowing for electron tomography experiments with ± 90 ° tilt angle range . the vacuum in the tem was 9 × 10 − 8 ton in the vicinity of the sample and the microscope was dry pumped . fig1 a ), b ) and c ) are tem images of substantially amorphous elk fibrils , that are obtained without a traditional zpp . in fig1 a ) traditional phase imaging ( applying a defocus ) is used to produce a phase image . tem images of the same sample in identical operating mode and focus except that hole - free thin films of carbon ( fig1 b )) and gold ( fig1 c )) are positioned at the selected area aperture ( which is conjugated with the back focal plane ) and no defocus is applied . it is noted that both fig1 b ) and fig1 c ) show comparatively higher contrast than fig1 a ). after inserting the hole - free thin film in the beam path , the ctf changes with time for an initial period of about 1 min ( gold ) to about 15 min ( carbon ). after the initial period the ctf is unchanged for an extended period of time . it was after this period that the images of fig1 b ), c ) were taken . the significance of using gold thin film material is that gold films are believed to be good conductors and surface layers of contaminants are rarely observed . nonetheless surface contaminants provide one possible explanation for the ctf observed , and may have a role in producing this phenomenon . it is even possible that surface contaminants are the primary reason for the consequent charging by the incident electron beam due to secondary electron emission . the change in defocus that appears to recur in all comparisons of images taken using defocus and those taken with various hole - free thin films , demonstrates that there is a phenomenon ( herein called ‘ charging ’) at work that changes the ctf of the microscope , as if a defocus and an additional phase shift at q = 0 were applied . applicant concludes that charging can be used productively to effect phase contrast imaging on hole - free thin films , as demonstrated by fig1 b ), c ), or further as a correction in amorphous thin film zpps ( with hole ), or other zpps that are subject to charging . fig2 a ), b ), and c ) show phase contrast transfer functions ( ctf ), i . e . the power spectra of the images of the elk fibrils taken with defocus phase contrast ( fig1 a )), a hole - free carbon thin film ( fig1 b )), and with a hole - free gold thin film phase plate ( fig1 c )), respectively . similarly , fig2 a ) is the ctf of the tem with the phase plate retracted from the electron beam , and a defocus of about 1000 nm applied . the ctfs ( fig2 ) depict the modulation of the contrast transfer from phase to amplitude ( phase - amplitude mixing ). the bright bands near the centres of the ctfs ( marked by arrows ) represent a transfer band near q = 0 . the presence of this band demonstrates that the technique provides improved contrast transfer at low spatial frequencies , resulting in enhancement of image contrast . the amount of contrast enhancement can be quantified , and it is about two to three times greater when using hole - free thin films , compared to images taken with defocus only . the deviations of the ctf from circular shape at high spatial frequencies ( further from the origin of the graph ) in fig2 ( as well as in fig3 c ) ( no zpp ) and fig3 d ) ( traditional zpp )) may be due to sample drift , and / or that charging may not exhibit radial symmetry possibly due to defects in the film or from non - radial symmetry beam current distribution at the back focal plane . the effect is similar to astigmatism and affects high spatial frequencies rather than low spatial frequencies . applicants have produced and tested single - layer thin films that are π / 2 - thick carbon and tested uniform aluminum film and gold film of thickness far exceeding π / 2 . even relatively thick gold films exhibit ctf changes that we attribute to charging . furthermore , it is shown that prior art zpps with a through hole are subject to charging . applicants have tested zpps where a hole was fabricated in a single - layer carbon and 3 - layer carbon - chromium - carbon stacked thin film . the zpps in these experiments were made either of electron - beam evaporated carbon or a multilayer consisting of carbon , chromium and carbon inducing ½π phase shift in each layer , ( totaling 3 / 2π for the multilayer ). the chromium layer is intended to increase conductivity of the zpp to reduce charging . in both cases the technique described above was used for deposition . in general , thin film zpps can be formed of carbon with a hole about 0 . 1 μm to about 2 μm in diameter . for the present experiments with pre - fabricated hole in the zpp ( the traditional set up ) the hole diameter in our zpps was between 100 nm and about 1 . 5 μm . the desired dimensions of the zpp hardware depend on the focal length f of the objective lens and the illumination condition of the tem . fig4 is a schematic illustration of the multi - layer zpp in accordance with an embodiment of the invention . fig4 a ) shows a cross - section of a three - layer zpp . the 3 - layer zpp was fabricated in a multiple step process . first a self - supporting carbon film π / 2 - thick was deposited onto a frame of selected area aperture , then a chromium film π / 2 - thick was deposited onto said carbon film and a large hole was drilled using a focused ion beam . then a carbon film π / 2 - thick covering the large hole in chromium + carbon bilayer was floated onto the bilayer . a hole was bored in the single carbon film in the centre of the large hole in the single carbon film in the centre of the large hole in the bilayer using the focused ion beam and a hole was bored in the 3 - layer film at a location distant from the carbon single layer . fig4 b ) is a schematic illustration of a top view of the traditional zpp in use , showing the geometrical condition at the back focal plane . the unscattered beam passes through the centre of the zpp , whereas scattered beams pass outside the centre of the zpp . the radial distance r varies inversely to the scattering vector q , which in turn corresponds to a spatial frequency transferred by the tem , because zpp hardware is placed in the back focal plane of the tem objective lens or plane a conjugate thereto . the radial distance at the zpp plane and the scattering vector are related by the effective camera length , determined by the optical parameters , such as focal length of lens above the zpp , at the plane of the zpp . fig3 a ) is a tem image of multiwall carbon nanotubes ( mwcnt ) with crystalline pt / ru nanoparticles imaged by phase imaging by defocusing . fig3 b ) shows the same sample imaged using zpp ( π / 2 thick carbon ) with a fib prefabricated hole . fig3 c ) shows a ctf from the support film area marked by a pale square in fig3 a ), and fig3 d ) shows a ctf from the support film area marked by the pale square in fig3 b ). the defocus for the image acquired without zpp in fig3 a ) and the corresponding ctf in fig3 c ) was about 1000 nm . the ( fig3 b ) image was obtained by setting a gaussian focus before the zpp was inserted , followed by insertion of amorphous carbon thin film zpp . the focus of objective lens ( ol ) and objective minilens were not changed , and care was taken to ensure that the zpp was in the saa plane , and thus was conjugated with the back focal plane of the ol ( as depicted in fig7 ). a change of defocus with insertion of zpp induced fresnel fringes ( profile shown in inset graph of fig3 b )). consequently it was concluded that the contrast fast oscillations in the contrast transfer function ( ctf ) in fig3 d ) are associated with the zpp , rather than any change of the lens of the microscope . a change of apparent defocus is therefore evident when a zpp with a hole is inserted into the tem . such a spontaneous change in focus is consistent with charging of the zpp hardware . fig3 c ) shows the ctf of the tem image using defocus , while fig3 d shows a ctf of a microscope with fib - prefabricated hole in a carbon thin film zpp . the band marked by an arrow in fig3 d can not be attributed to defocus , lens spherical aberration , or presence of π / 2 phase shift from the zpp . the ctf shown in fig3 d ), cannot be fitted to the expected ctf using the defocus z and spherical aberration c s . in particular , the transfer band marked by an arrow in fig3 d ) distinguishes the measured ctf from the ctf of a microscope equipped with an ideal π / 2 - thick zpp . this might be attributed to contamination buildup and hole drilling in the zpp material , and charging of the zpp . our measurement of thickness of a zpp film by energy - filtering tem after extensive use , rules out contamination and hole drilling under the used conditions . from our observation of the ctf , a bright field image of the zpp , and point projection holograms of the zpp under electron beam irradiation , the most likely explanation appears to be charging of the carbon film of the zpp . fig5 shows the imaging conditions at reciprocal space plane for the image shown in fig3 b ). the fib fabricated hole is marked by a circle in the upper left corner while bragg reflections originating from 0 . 34 nm lattice of mwcnt are marked by an arrow . in this case ( fig3 b ) and fig5 ) the zpp hardware was placed in selected area aperture plane and objective minilens was used to transfer the back focal plane of the objective lens to the selected area aperture plane that was conjugate with the back focal plane of the objective lens . fig5 shows that the diameter of the zpp hole was about 500 nm , and the angular width of the incident electron beam used in these experiments . the bragg spots marked by an arrow originate from 0 . 34 nm spacing of multiwall carbon nanotube allowing us to accurately calibrate the imaging conditions in reciprocal space . the convergence angle of the incident kohler illumination is about 40 μrad and the cut - on angle of the zpp hole is about 100 μrad corresponding to phase shifting of spatial frequencies above 0 . 4 nm − 1 or 24 nm in real space . consequently , applicant concludes that this new knowledge can be productively applied in transmission electron microscopy as follows : a method of preparing a transmission electron microscope for phase contrast imaging may involve placing a hole - free thin film at a back focal plane ( bfp ) or a plane conjugate thereto ; focusing an electron beam of the transmission electron microscope for phase contrast imaging for an initial period of time during which charging and dissipation rates on the thin film are not in balance ; and then performing phase contrast imaging once the charging and dissipation rates stabilize . it is possible that during the initial period the ctf changes involves changes in surface contamination layer thickness induced by the incident electron beam , in addition to the charging . a tem is provided comprising an electron beam source , sample region , objective lens , and projection system wherein a hole - free amorphous or crystalline thin film is placed at a back focal plane or a conjugate plane thereof with the objective lens operated so that a scattered wave and unscattered wave are focused on different areas at the back focal plane . in the foregoing examples , we focused on amorphous thin - film type zpps , but the implications of charging to electrostatic - type zpps can be used to generate a control signal to the einzel or boersch lens to compensate for the charging . the smaller area of electrostatic - type zpps susceptible to charging , the broken radial symmetry of the zpp caused by the structure of the zpp , the large thickness of the conducting electrodes of the boersch lens and the significant thickness of boersch lens , which tends to stop primary electrons , are all factors that will change the charge balance in the device , in comparison with film type zpps . without wanting to be limited by the following theory in all aspects of this invention , applicant considers the charging to likely be produced by secondary emission of electrons . emission of secondary electrons ( se ) induced by an incident ( primary ) electron beam is a well established fact responsible for se images in scanning electron microscope ( sem ) and se mode of scanning tem ( stem ) [ 17 , 18 ]. the number of ses emitted per an incident primary electron ( the se yield ) varies with incident energy , incident angle and irradiated material but is always non - zero [ 18 ]. the emission of ses results in positive charge buildup on the irradiated object [ 19 , 20 ] such as zernike phase plate ( zpp ), the sample itself , an aperture , or polepiece and beam liner in a tem . the positive charge is compensated by induced current flowing from a ground electrode and driven by a positive potential developed due to se emission . eventually a steady state develops , and the emitted ses are fully compensated by induced current from ground electrode . the period it takes for a steady state to develop as well as the steady state itself can be affected by possible surface contaminants layers . a beam - induced surface contamination build up produces phase shift in addition to the charging . it is important to note that the steady state requires a gradient of electrostatic potential continuously driving the electric current from the ground electrode to the area where ses are generated by an incident primary beam [ 19 , 20 ]. the steady state can develop rapidly with small gradient of electrostatic field in good conductors , while a footprint of a charged area can remain for appreciable period of time after the primary beam was removed in poorer conductors [ 21 , 22 ]. the presence of a se emission - induced electrostatic potential described above leads to an electrostatic phase shift ( eps ) of the primary electrons in addition to phase shift induced by the mean inner potential ( mip ) of the zpp film including surface contaminants [ 23 ] or the electrostatic phase shift in electrostatic - type zpps . the electrostatic potential , and consequently the eps , can be significant , even for moderate charge densities resulting from se emission [ 20 ]. whatever the origins of the charging , a model of the effect of static charge on an object ( such as the zpp ) in the back focal plane on the contrast transfer function may be used . these models may not be accurate , and empirical and / or semi - empirical procedures may be chosen to compute the desired field strengths of electrostatic - type zpps , or to compute a desired thickness for an amorphous thin film zpp suited to desired operating conditions within a tem , or conversely for selecting operating conditions for an amorphous thin film zpp of a given thickness , with or without a hole of a given size . particularly the condenser settings can be controlled to empirically optimize the contrast directly observed on a digital camera located at the image plane of the microscope . charging of various objects in the beam path can have profound effect on the electron optics of a tem . all objects that emit se upon incidence of a primary electron are subject to some degree of charge redistribution leading to an electrostatic potential . in case of broad beam illumination at a sample plane of a tem , the effect of the electrostatic potential ( bias ) due to se emission adds a constant phase shift across the entire illuminated area leading to no observable effect ( it is the square of the amplitude that forms an image , a constant phase shift is therefore not observed ). for most biological samples the scattering is very weak and diffuse ( as opposed to strong bragg reflections observed in crystals ) leading to smooth ( i . e . not a step function ) current density profile at the back focal plane where the zpp is placed . in case there is a time - dependent component in the bias , a beehive effect [ 28 ] can be observed . when a bias is non - uniform ( and even more so when it acts in reciprocal space ) such as in the back focal plane of a lens , the charging may strongly affect the resulting image , which would explain the demonstrated phase contrast imaging on a hole - free zpp . in particular , a spatial frequency - dependent phase shift , as found in a charging zpp , can entirely dominate the contrast formation mechanism in a tem . in the case of zernike phase plates , an object located in high current density area in the back focal plane of a tem , it is considered possible that charging can be sufficient to generate a potential leading to significant alternation of electro - optical properties of the phase plate . for our particular example the calculated charge - induced phase shift near q = 0 the phase shift exceeds the π / 2 by nearly an order of magnitude . the applicant concludes from the profiles of the ctf in fig2 b ) and 2 c ) and fig3 d ) that using a thin - film zpp , the phase shift due to charging can entirely mask the π / 2 shift desirable for zernike contrast in tem [ 23 ]. it is common practice in tem and sem to coat a sample with thin film of carbon or other material to reduce charging of a non - conducting sample . it should be emphasized that the carbon or similar coating only reduces the charging , but does not eliminate the se and charging , as readily witnessed by ability to obtain se images from carbon - coated samples in stem and sem . charging can be readily observed when the microscope optics is set up for point projection holography ; a cross over is formed at a small distance above the sample and defocused image of the sample is projected onto a screen . although uniform ( long - range ) and time independent charge induced phase shift would be as difficult to detect in point projection set up as it is in off - axis holography and in bftem , instabilities in charging can be readily observed in point projection holography up as they induce minor sample jumps ( for large instabilities ). the charging of an object at an image plane ( sample ) due to se emission can be responsible for apparent increase of mean inner potential of nanoparticles with decreasing particle size [ 32 ]. to image an object at low irradiation dose it is most important to correctly transfer the lowest spatial frequencies [ 16 ], i . e . near q = 0 . adjusting the phase shift of the direct beam ( near q = 0 ) to a phase shift π / 2 + nπ ( n is an integer ) with respect to ( elastically ) scattered beams should increase contrast and therefore decrease the irradiation dose needed for desired signal - to - noise ratio in the final image . since the se emission and charging and the resulting phase shift is proportional to beam current density j ( r ) in the back focal plane , it can be in principle optimized by adjusting the condenser setting while observing contrast in the image . in accordance with an embodiment of the invention , a method is provided for tem imaging . the method involves inserting a zpp ( of any type ) into a tem at a back focal plane of the objective lens or a plane conjugate thereto ; applying objective lens fields to focus an electron beam such that unscattered waves travel through a localized region of the zpp ( referred to as the centre of the zpp ), and scattered waves pass through a region surrounding the centre of the zpp ; and adjusting condenser settings of the tem to optimize contrast within the image . for example , a suitable thin ( carbon ) film can be placed in one of the standard objective aperture discs , inserted in the beam path at a nearly arbitrary position in the direction perpendicular to the electron beam , and then current density can be adjusted by adjusting the condenser lens to improve contrast and the ctf . the contrast and ctf may be observed as an intensity profile of image and simultaneously as a live fast fourier transform ( fft ) of the image respectively . this is an important advantage in terms of aligning and positioning the thin film in the tem . another way that a hole - free thin film can be prepared for phase contrast imaging , in situ in a tem , is for the electron beam to be focused on the thin film while no sample is placed in the beam , and operated at a high beam current so as to bore a hole in the thin film , to effectively form a standard zpp . the intense electron beam naturally defines the center of the tem , the position of the hole produced will therefore be aligned with the electron beam during operation . furthermore , under suitable conditions , the hole size can be well matched to the incident beam angular width and the position of the hole is determined by the position of the incident beam in the reciprocal ( q ) space . in fact , the match of the fidelity of the optical conditions , such as appropriate hole size ( cut - on spatial frequency q ) to beam convergence angle and positioning , can exceed fabrication limits of focused ion beam techniques for producing thin - film zpps . this is an important advantage in terms of aligning and positioning the thin film in the tem . the possibility to fabricate the hole in situ eliminates the need for focused ion beam boring , a fabrication step that is known to induce a ga - ion contamination of the zpp . no precision hardware is needed for positioning the zpp device relative to the incident beam . there are several physical mechanisms that may contribute to drilling of a hole under an intense electron beam [ 2 ] such as : radiolysis , electron stimulated desorption and electron beam sputtering or electron beam induced film etching by residual water vapor in the column . it appears that electron beam sputtering , which involves momentum transfer from the incident electron to the sample ( or thin film ) atoms , is the most suitable for practical reasons [ 27 ] providing a the zpp film is suitably selected that is subject to electron beam sputtering . a suitable material that is subject to electron beam sputtering has to have a sputtering threshold below the incident electron energy ( 200 kev in our case ) and a sputtering cross - section sufficiently high to drill a hole within a reasonable time through the entire film thickness . most light elements with atomic number less than about 20 have sputtering thresholds below 300 kv and are therefore suitable material for in situ fabricated zpp in standard tems . for example amorphous carbon has a sputtering threshold around 100 kv and an electron sputtering cross - section in the order of σ = 8 × 10 − 23 cm 2 at 200 kev incident electron energy . [ 27 ] the removal rate of the material from the zpp film can be calculated [ 27 ] according to the equation r = σdj / e , where r is the removal rate of the film material in monolayers per second , j is the incident current density ( constant over the drilled area ) and e is the electron charge in coulombs . for typical conditions that may be suitable for hole fabrication the incident current i = 1 na , desired hole diameter d h = 500 nm leading to j = 5 × 10 3 a / cm 2 at the back focal plane of the objective lens , and film thickness t = 22 nm ( about π / 2 at 200 kv ) the hole drilling time is about 30 to 60 s . the rate of material removal is additionally affected by the presence of water vapour that enhances drilling rate , or by presence of contaminants that reduce the rate of drilling . since the current used for typical low dose imaging is significantly lower than the above - mentioned value ( typically below 0 . 1 na ), the in situ drilled hole can be used for at least 5 minutes to 10 minutes if all the incident current was further expanding the hole size . however , during the imaging ( after a hole was drilled through the thin film ), a significant fraction ( over 90 % for typical tem sample ) of the beam current passes through the hole in the zpp film rather than sputtering the zpp film further . with this in mind , the in situ drilled zpp device can be used for several hours without significant change . to demonstrate the possibility of hole drilling , several experiments were performed . fig6 a ) shows an image of an in situ drilled hole in a thin carbon film having a − 500 nm hole drilled by a 200 kev lab 6 instrument ( jeol 2010 ). the carbon film was placed at the sample plane in this experiment . a standard positioning mechanism ( usually used at the selected area aperture or objective aperture ) such as stepper motor driven selected area aperture mechanism in a jeol2200 fs or manually positioned aperture assemblies in older microscopes provide sufficient drift stability for long term ( hours ) operation of the zpp with typical dimensions of zpp hole ( 200 nm to a few micrometers ). fig6 b ) is a graph and table showing typical values of boring at a sample plane of a carbon thin film . following the method described above , the hole diameter ( i . e . cut - on angle ) will match the angular width of the incident illumination . since the sample is not present in the beam path during the hole drilling step , the hole should be an accurate representation of the incident illumination shape in reciprocal ( q ) space . when a sample is inserted in the beam , the electrons scattered outside the angular width of the incident beam will be phase shifted by the in situ drilled zpp device . a typical sequence for phase contrast imaging using an in situ drilled amorphous thin film is as follows : align microscope and set microscope magnification ; set the condenser lens for desired imaging condition , such as kohler illumination , and desired current density ; find a hole in the sample , and align the hole in the sample with the electron beam while passing through a hole in the sample ; insert a zpp film in the back focal plane or in a conjugate plane thereof : wait to for the incident beam to drill a hole in the zpp film ; move the sample from the hole to an area of interest , and start imaging the sample . as noted above , applicant has found advantages in placing a zpp or hole - free thin film at conjugate plane to the back focal plane produced by a cross - over induced by an objective minilens after the objective lens . in accordance with the prior art , the zpp is usually placed in the back focal plane ( bfp ) of the objective lens or a special transfer lens is incorporated in the microscope column to accommodate the zpp . fig7 shows ray path diagrams for a tem having a zpp or hole - free thin film at the saa . in fig7 a ) the objective minilens ( om ) is turned on for phase contrast imaging , and in fig7 b ) the objective minilens turned off to realize a standard phase contrast , or diffraction contrast imaging mode . as shown in fig7 a ), a tem equipped with an objective minilens , permits placement of the zpp or hole - free thin film at a selected - area aperture ( saa ) plane , such that the bfp is transferred to the saa plane by suitable excitation of the objective minilens [ 23 ] making it convenient to install the zpp or hole - free thin film . blue crossover corresponds to diffraction pattern and red cross over corresponds to an image plane . the intermediate lens ( post zpp lens ) is then used to focus the sample while the objective lens and objective minilens and condenser lens system stay unchanged . the physical space available at the saa plane is significantly larger than the space available at the back focal plane of the ol : the space at the saa plane can be easily 10 mm along the beam path as well as in the plane perpendicular to the electron beam . thus the use of objective minilens ( om ) brings additional degrees of freedom to ensure that the zpp hardware is in a plane accurately conjugated to bfp of the objective lens . in the case of a zpp placed directly in the back focal plane , only the sample height and the focal length of the ol can be varied . when the om is used an additional variable , the focal length of the om can be used to optimize the electro - optical conditions . an important advantage can be obtained by focusing with a lens below the zpp or hole - free thin film , since any lens below the zpp device does not affect the position of the back focal plane at the zpp device . in the traditional operation the position of the conjugate plane of the objective lens moves along the optical axis as the objective lens ( or objective minilens ) is focused on the sample . the movement of the diffraction plane along optical axis can result in a change of alignment of the zpp or hole - free thin film with respect to the conjugate of the bfp . using condenser lenses to focus the back focal plane onto the zpp device results in limited control over illumination conditions . applicants have demonstrated that a hole - free thin film can be prepared for phase contrast imaging in situ within a tem . phase plates with prefabricated holes , with in situ fabricated holes , and without any hole are subject to charging that provides a contrast transfer function that can be leveraged for phase contrast imaging . additionally the hole - free thin film may be bored in situ to produce a standard thin - film type zpp , without the difficulties associated with fabrication and alignment in the prior art . other advantages that are inherent to the structure are obvious to one skilled in the art . the embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed . variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims . 1 f . hosokawa , k . nagayama , and r . danev , u . s . pat . no . 6 , 744 , 048 ( 2004 ). 2 r . f . egerton , p . li , and m . malac , micron 35 , 399 ( 2004 ). 3 m . malac , m . beleggia , r . egerton , and y . zhu , ultramicroscopy 107 , 40 ( 2007 ). 7 r . danev , r . glaeser , and k . nagayama , ultramicroscopy 109 , 312 ( 2009 ). 10 t . masumoto , n . osakabe , and a . tonomura , u . s . pat . no . 5 , 814 , 815 ( 1998 ). 11 e . majorovits , b . barton , k . schultheis , f . perez - 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