Ultramicroscopy 150 (2015) 44–53

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Transmission environmental scanning electron microscope with scintillation gaseous detection device Gerasimos Danilatos a,n, Mary Kollia b, Vassileios Dracopoulos c a

ESEM Research Laboratory, 28 Wallis Parade, North Bondi, NSW 2026, Australia Laboratory of Electron Microscopy and Microanalysis, School of Natural Sciences, University of Patras, GR-26504 Patras, Greece c Foundation for Research & Technology-Hellas (FORTH), Institute of Chemical Engineering Sciences (ICE-HT), Stadiou Str., Platani P.O.Box 1414, GR-26504 Patras, Greece b

art ic l e i nf o

a b s t r a c t

Article history: Received 2 September 2014 Received in revised form 3 November 2014 Accepted 10 November 2014 Available online 5 December 2014

A transmission environmental scanning electron microscope with use of a scintillation gaseous detection device has been implemented. This corresponds to a transmission scanning electron microscope but with addition of a gaseous environment acting both as environmental and detection medium. A commercial type of low vacuum machine has been employed together with appropriate modifications to the detection configuration. This involves controlled screening of various emitted signals in conjunction with a scintillation gaseous detection device already provided with the machine for regular surface imaging. Dark field and bright field imaging has been obtained along with other detection conditions. With a progressive series of modifications and tests, the theory and practice of a novel type of microscopy is briefly shown now ushering further significant improvements and developments in electron microscopy as a whole. & 2014 Elsevier B.V. All rights reserved.

Keywords: ESEM TESEM ESTEM wet-ESEM GDD Transmission environmental scanning electron microscope Dark field Bright field Gaseous detection device Carbon nanotubes

1. Introduction The possibility, practice and theory of imaging with a scanning electron microscope (SEM) in transmission mode has been presented in a recent review by Klein et al. [20] covering the entire period since von Ardenne [28,27] introduced the SEM. Thus, a conventional SEM can also be used as transmission SEM (TSEM), i.e. as a scanning transmission electron microscope (STEM) but in the generally low accelerating voltages employed in SEM. Correspondingly, an environmental scanning electron microscope (ESEM) can also be used as transmission ESEM (TESEM), i.e. as an environmental scanning transmission electron microscope (ESTEM) but in the generally low accelerating voltages employed in ESEM. It is important to differentiate the terminology and acronyms used correctly, in order to avoid confusion in future electron microscopy practice. First of all, a conventional STEM has a different electron optics architecture from a SEM. Furthermore, if we maintained the established term STEM and, hence, ESTEM n

Corresponding author. E-mail address: [email protected] (G. Danilatos).

http://dx.doi.org/10.1016/j.ultramic.2014.11.010 0304-3991/& 2014 Elsevier B.V. All rights reserved.

universally, from a practical point of view we would also have to distinguish the conventional high voltage (HV) operation as ESTEM-HV and the lower voltage (LV) ESEM operation as ESTEM-LV. However, LV has been used also in a different context of low voltage in LV-SEM [22], also low vacuum SEM by some manufacturers, and so it seems more practically suited to settle with the Klein et al. term of TSEM, which also implies a SEM electron optics column. The latter authors presented similar arguments in choosing TSEM among a variety of previously used terms. Accordingly, we now simply extend the same approach to TESEM. Clearly, the latter term is more general and inclusive also of the “wet-STEM”, which denotes wet specimens only. The use of an ESEM in TESEM mode has already been reported as a wet-SEM by Bogner et al. [2,1], who used an annular BSE detector below the specimen to obtain dark field (DF) images and applied it to the study of various particles in a liquid water phase. This was followed with work by Maraloiu et al. [21], who applied the same technique to the study of magnetic nanovectors. Staniewicz et al. [26] also applied two solid state segment detectors below the specimen, one serving as dark field and the other one as bright field (BF) imaging for the study of bacteria. These and other

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similar publications have employed only a solid state detector below the specimen to directly detect the DF and BF signals in the conventional manner, but no attempt has been reported yet in the use of the gaseous detection device (GDD) for TESEM, or ESTEM as prescribed by Danilatos [9, pp. 89, 90] and in associated detailed patent applications and specifications during the last decade. In this report, we present a novel approach and practice for a TESEM with the use of an existing commercial machine employing a scintillation gaseous detection device. We explain the microscopy mechanisms involved together with the practical steps taken in order to demonstrate primarily the “proof of principle” without attempting to determine the optimum conditions of operation. Even so, some high resolution imaging of both bright and dark field transmission electron microscopy in gas together with ancillary images for comparison is provided.

2. Method, materials and apparatus 2.1. Gaseous detection device There has been wide use of a commercial (FEI) type of detector called “gaseous secondary electron detector” (GSED). It is important to realize that this detector is only a partial application of the more general concept of a gaseous detection device (GDD), whereby the gas is used as a detection medium, i.e. simultaneously with its use as an environmental medium. By this concept, all signals interacting with the gas in some way can be detected by sensing, counting or collecting the products of signal–gas interactions as originally disclosed by Danilatos [6]. Among the multiplicity of such products, electron–ion pairs (ionization) and photons (excitation) are of immediate practical importance and have already been used for ESEM imaging. A GDD based on ionization has been denoted as ionization-GDD in distinction from a scintillation-GDD [7,9]. FEI company has employed particular forms of the ionization-GDD type, whilst Zeiss company has employed a particular form of the scintillation-GDD type. The GDD is akin to what is known as an immersion detector in nuclear physics technology, whereby the radiation source is

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located inside the detector. In our case, the signal producing source at the beam specimen interaction volume is enveloped by the environmental medium acting as detector at the same time. It can be said that the gas coexists in harmony with the detector and not in opposition, as it happens with the Everhart–Thornley (ET) detector that requires high vacuum. 2.2. Microscope In this work, we use a LEO SUPRA 35VP FESEM commercial brand of an ESEM. This machine uses the scintillation-GDD type of detector together with a high resolution field emission electron gun and a dual electrostatic and magnetic objective lens (Gemini type). It operates in two regimes: (a) In high vacuum mode it uses the dual objective lens with an in-lens secondary electron (SE) detector, or with a conventional ET detector. (b) In the so-called “variable pressure” (VP) mode, nitrogen gas up to a maximum pressure of 133 Pa is introduced as a scintillation detection medium in conjunction with a photomultiplier (PMT) at the same time as an environmental medium for the examination of insulating specimens. A light pipe is placed in front of the PMT to guide light (gaseous photons) from a region in the neighborhood of the specimen. These photons are produced generally by SE but also by backscattered electrons (BSE) all amplified in an electron avalanche generated in the electric field formed by a biased electrode close to the PMT. This system has been termed “variable pressure secondary electron” (VPSE) detector. The relative intensity of photons produced by the BSE and SE is a function of gas pressure and geometry of the system used. In short, this detector constitutes a scintillation proportional amplifier in the ESEM, the various parameters of which have been quantitatively analyzed in the original description by Danilatos [9]; reference to that work will aid in the better understanding of the current developments. In the gaseous mode of operation, the electrostatic lens is automatically switched off to avoid a catastrophic discharge by the high voltage (kV range) applied to it. The resolving power of this instrument is 1.5 nm at 20 kV for in vacuum operation with ET and in-lens detectors according to the data sheets provided by the manufacturer.

Fig. 1. (a) LEO SUPRA 35VP FESEM with thin specimen transmitted by an electron beam. Various fractions of signals above and below the specimen reach the photomultiplier (PMT). (b) Imaging according to (a) with PtCo on CNTs on top of carbon film: increased noise and beam damage rasters created at higher magnifications are present at moderate magnification of 18.82 kX, at 3.4 min.

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The above microscope operation is better explained with reference to the diagram in Fig. 1a. The exact dimensions have been provided in two previous reports using the same machine [5,17], where the gas flow properties have been mapped by use of the direct simulation Monte Carlo (DSMC) method. The electron beam transmittance of decaying intensity has also been plotted along the electron beam axis for various pressures and accelerating voltages both for the unmodified machine and for a proposed improvement of it. Because the electron beam travels a relatively long distance in the gas before it enters the specimen chamber (about 9 mm), it undergoes severe and unnecessary losses restricting the lowest accelerating beam voltage possible. Its performance was evaluated by an introduced figure of merit [11] and the main parameters involved have been properly defined in a recent report [13]. Thus, we note that the environmental distance (e_d) between A and B is greater than the working distance (w_d) measured from the lens pole-piece. The final pole-piece of the electromagnetic lens has a bore diameter of 5 mm, inside which fits the column liner (also electrostatic lens) with a diaphragm (A) of 1 mm acting as a pressure limiting aperture (PLA). By such means, while the pressure p0 in the specimen chamber can be maintained up to 133 Pa with nitrogen gas (i.e. a limit set by the manufacturer), the pressure p1 above the PLA is a few orders of magnitude less. Further details of the machine are described together with various detection configurations below. 2.3. Detection configurations Unmodified instrument: Various signals are emitted and detected first without any modification according to Fig. 1a. This instrument is normally used for the examination of the surface of bulk specimens as in any conventional SEM but also with addition of a gaseous environment. If we now use a thin specimen traversed by the beam (instead of a bulk one), we encounter additional multiple signals that may contribute to the formation of an image if no special measures are implemented. The primary beam first generates ionization and scintillation in the initial path AB (¼ e_d) of the gaseous layer above the specimen surface. Electrons removed from the beam produce photons in the gas along the beam path but also away from the beam wherever they can further interact with the gas molecules by additional collisions. Electrons can also undergo multiple scattering between various walls and between gas molecules in every possible combination resulting in photon production almost everywhere. Clearly, although the PMT aims to preferentially detect light from the region immediately above the specimen specifically associated with secondary electrons, it turns out to receive photons from almost everywhere in the chamber with variable intensity. Corresponding to the travel distance AB, the beam generates a steady (uniform) signal in the form of light, a significant fraction of which, denoted by S(AB), reaches the PMT. This fraction does not carry any information from the specimen and simply adds background noise to the image. The travel distance BC is inside a specimen section with B and C being the entry to and exit point from it. Signals are generated along this entire distance/volume between B and C and emitted in all directions but not all of them manage to reach the PMT. For this report, let us use the symbol S(B) to denote all signals escaping above the specimen that manage to reach the PMT and the symbol S(C) to denote all signals emitted below the specimen that manage to reach the PMT. B and C are only symbolic “points” in this notation and should not be perceived as restricting the precise location of signal emission. Only a small fraction of the total signal is dissipated inside the specimen due to its usually small thickness. Each of these two useful fractions of signals eventually produces photons via which they are detected by the PMT and contribute to the formation of an image. Associated with S(B) are all types of SE

and BSE signals, whilst associated with S(C) are the forwardly transmitted-and-scattered electrons that are known to form the dark field (DF) contrast in STEM. In addition, we also have all the secondary electrons emitted from the bottom surface of the specimen by the action of the exiting DF electrons (DFE). We may denote the latter SE component by SE-T (i.e. secondary electrons transmitted) and, whilst directly associated with the DF signal, it does not contribute to the classical way of DF imaging in vacuum. The GDD now allows both components (DFE and SE-T) to take part in image formation. However, the relative contribution to the total DF contrast by each of these two signal mechanisms is the subject of further investigation, but suffices it to state that we now have novel means for DF formation in a TESEM. Finally, there are the bright field electrons (BFE), i.e. the fraction of primary beam that passes through the specimen without scattering outside the beam probe aperture and traveling through the remaining gaseous distance CX before it terminates at some point X, usually on a chamber wall especially at the low gas pressure of operation. The BFE intensity is modulated by the losses the beam undergoes in the form of DF signal, resulting in the well known reversal of contrast. Again, via a conversion to gaseous photons, a significant fraction of BF signal denoted by S(CX) reaches the PMT. The properties of both DF and BF signals depend on specimen nature, thickness and accelerating voltage, and vary according to established rules in conventional STEM, like the scattering angle of dark field electrons, probe aperture and detector aperture [24]. The novel aspects herewith are that (a) the SE-T generated from the bottom surface by the exiting DF may also join in the generation of the final DF image, (b) all signals are immediately converted to gaseous ionization and scintillation and finally can be counted by appropriate means, like by a PMT in the present machine, and (c) the electron beam scattering effects are considered along the entire travel distance of the beam through both specimen and gas system in tandem. The signals above the specimen include all types of secondary electrons SE(I, II, III, IV) and BSE [23], which further interact with the gas molecules to generate electrons and photons. The multiplicity of all such signals and more in their signal–gas interactions has been analyzed in detail and a rational scheme of terminology has been proposed in order to understand and apply the gaseous detection device in the best possible manner in pages 36–42 by Danilatos [9]. Furthermore, we need to elaborate on the SE-T signal and its origin and how it could affect both BR and DF imaging. In general, the total signal S collected by the PMT in the unmodified system may be formally stated to be:

S = S(AB) + S(B) + S(C) + S(CX)

(1)

Apparently, imaging under these conditions is possible but not with best resolution and signal-to-noise-ratio (SNR): The background (white) noise S(AB) is relatively large in the present instrument and the available contrast is further diminished as various signals cancel each other to varying degrees depending on the shadowing effects of intervening walls, like between the point of origin of DF or BF and the position of PMT. In addition, the entire specimen interaction volume (depending on the total specimenfilm thickness) lowers the ultimate resolution possible. To counter all this, a series of steps has been undertaken to separate the most useful signals bearing the highest contrast and resolution as outlined below. Various tests have also resulted in some unique imaging techniques, some of which deserve a separate report. Here, we select only information and results that are sufficient for a “proof of principle” of a novel TESEM fully equipped with both bright and dark field imaging capabilities bearing high contrast and resolution.

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Fig. 2. LEO SUPRA 35VP FESEM operating as TESEM with bright field imaging (a) and dark field imaging (b) capability. An aluminum tubular shield above the specimen effectively eliminates all signals above the specimen. An inverted carbon Faraday cup below the specimen in (a) effectively eliminates all the dark field electrons except those associated with bright field contrast. A carbon Faraday cup at some distance below the specimen in (b) effectively eliminates the bright field electrons except those associated with dark field contrast.

Bright field: Based on the above description of the system, suitable electron and photon shields or traps have been made around the specimen as can be seen first in Fig. 2a. An upper shield made of aluminum in the form of a tube with 1.8 mm inside diameter, 2.4 mm outside diameter, and 4.8 mm inside height (made up of 4.1 mm outside tube height on a 0.7 mm thickness disk) is placed above the specimen and inserted in the bore (5 mm diameter) of the objective lens of the machine. Testing showed that an insertion depth of more than 1.5 mm inside the lens bore is sufficient to shield all light produced above the specimen from being detected by the PMT. The addition of a lower shield made of carbon with a small hole of 0.7 mm diameter at 4 mm distance below the specimen effectively traps inside its cavity in the distance around CD the electrons transmitted-and-scattered out of the beam in the specimen distance BC together with all associated photons and SE-Ts. A thin disk between the upper and the lower shield holds the specimen securely in place, while all pieces are made concentric with the same outside diameter for ease of centering along the beam axis. Adhesive conductive tape is used to hold all pieces together and the assembly is mounted on the specimen stage in the usual manner. By this device, all photons and electrons produced along the entire beam path are screened out except for those transmitted within a narrow aperture around the axis of the transmitted electron beam in the travel distance DX. This last signal is associated with bright field imaging as in conventional electron microscopy. The difference is that, instead of an electron detector placed directly in the path of the transmitted beam, the same beam is allowed to dissipate its energy in the environmental gas of the specimen chamber directly producing electron–ion pairs and photons, which are further multiplied by an ensuing electron and photon avalanche towards the PMT of the given machine, as mentioned earlier. For simplicity and to show the new features of the work, the electron beam is shown by a straight line, while it actually forms a sharp cone with a given “probe aperture”. The BF electrons may contain both completely unscattered beam electrons and some scattered electrons that fall within the small angle defined by the screening aperture chosen (corresponding to a “detector aperture”

in STEM). There is a case here for both such events since, for example, the mean free path of 30 kV electrons in carbon is 20 nm [25], which means that about 1/3 of beam electrons are transmitted without any scattering through a specimen of this thickness, as can be easily shown. The fraction of unscattered electrons decreases exponentially with mass thickness while the probe spot gradually broadens by the time it exits the specimen. Whilst in conventional STEM the optimum condition is achieved by making the detector aperture about equal to the beam probe aperture, in the present system we have to make the detector aperture sufficiently larger to allow also for the scanned raster size to fit within the detector aperture, but this may vary according to magnification used and subject to later technological optimization advancements. Now, to the extent that the collected BF electrons contain a scattered fraction from the specimen (as it happens by default), the corresponding fraction of SE-T are lumped together with the remainder fraction of SE-T generated by the DFE. These two SE-T fractions may be indistinguishable, as their spatial distributions are closely overlapping due to the small specimen thickness. The SE-T are also a mix of probably indistinguishable SEIT and SE-IIT electrons, i.e. SE-I liberated from a single collision and SE-II liberated from more collisions of the primary electrons within the escape depth of SE from the bottom of the specimen. The distinction among various types of SE becomes somehow blurred in the case of very thin specimens with thickness less that twice the escape depth of the SE. Specimen thickness and mean free path also interplay in the determination of transmitted primary beam fraction with totally unscattered electrons and in the determination of amount of beam broadening. For a practical purpose, no SE-Ts contribute to the BF image formation according to Fig. 2a, because they are trapped inside the Faraday cavity. However, they may vary the contrast in DF imaging according to the following detection configuration. Dark field: By re-arranging/modifying the shields and traps in the same machine as is shown in Fig. 2b, we can similarly obtain dark field imaging corresponding to conventional scanning transmission electron microscopy. The upper shield and the specimen holder remain the same, but now an effective Faraday cage

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made by a 0.5 mm diameter hole (about 4 mm deep) on an aluminum cantilever 2 mm wide is placed at 2 mm below the specimen. It is held in place via an appropriate spacer that allows more than 270° free view (i.e. unobstructed) normal to the beam axis. By such means and under the appropriate choice of gas pressure, aperture size and distance (here, 25 Pa, 0.5 mm and 2 mm, correspondingly), most of the bright field electrons are eliminated by the Faraday cup below without many gas interactions in the intervening short distance CD, i.e the bright field signal is effectively suppressed. On the contrary, the majority of forwardly scattered electrons remain free to interact with the gaseous medium outside the Faraday cup. The latter electrons are associated with dark field contrast and are detected by the given PMT after they undergo collisions with the gaseous medium and between walls, conversion to photons and finally become multiplied in a photon avalanche towards the biased electrode of the PMT. The sum total of all associated SE-T may now be detected via the gaseous medium, which is the subject of further investigation. In support of the above claim that the BF electrons do not produce any significant number of interactions with the gas in the intervening distance CD, we can do the following simple calculation. The average number of scattering events m of primary electrons in the gas is given by

m=σ

p0 kT

(CD)

(2)

where s is the scattering cross-section, p0 the gas pressure, k Boltzmann's constant and T the absolute temperature [11]. Substituting the parameter values given above and taking T ¼293 K and s ¼ 1.59E  21 m2 at 30 kV for nitrogen [14], we obtain m ¼0.0196, which is extremely small, i.e. yielding a beam transmission factor of 0.98 from

f = exp( − m)

(3)

In other words, only 2% of the BFEs undergo scattering events in CD. Corresponding to the diagram in Fig. 2a and for better understanding, we provide a side overview of the BF assembly and specimen chamber in Fig. 3a by use of the available inspection camera of the instrument. The entire assembly is withdrawn below the lens whereby we see the upper and the lower shield in a continuous configuration. Similarly, corresponding to the diagram in Fig. 2b, we provide a side overview of the DF assembly in Fig. 3b. Again, the entire assembly is withdrawn below the lens and is tilted in order to make visible the lower Faraday cup shield (cantilever) separated by some spacer distance below the specimen

disk holder with upper aluminum shield; the viewing camera has a fixed position while the specimen disk partly obscures the lower shield in the horizontal position. Furthermore, corresponding to the diagram in Fig. 2a, we provide a micrograph overview of the BF assembly in Fig. 4a. The copper grid is in focus, whilst both the rim of the upper aluminum shield and the hole in the lower carbon shield are out of focus. Similarly, corresponding to the diagram in Fig. 2b, we provide a micrograph overview of the DF assembly in Fig. 4b. The copper grid is in focus with carbon film showing breakages at the end of multiple tests. The out of focus dark hole in the center is on the aluminum cantilever below the specimen, over which we obtain DF imaging; the upper aluminum shield shows its top circular bright rim out of focus. The assemblies are placed at sufficient distance below the pole-piece to allow imaging with the standard VPSE detector of the microscope. It should be noted that focusing is not allowed above the pole-piece (by manufacturer restriction) and the upper shield becomes invisible when the BF and DF modes are in operation. The size of the bright and dark holes seen at the center in both cases is the detector aperture and defines the field of view at present. Neither of the shown assemblies has been designed for optimum detection and operation yet. As starting work, the maximum accelerating voltage available on this machine, namely 30 kV has been used with nitrogen gas at 25 Pa. The electrode bias at the PMT has been fixed at 300 V. The working distance is kept constant at w_d ¼2.8 mm (unless otherwise indicated), so that the environmental distance remains at e_d ¼11.8 mm. For a better understanding of the current developments and more exact details of the geometry of the machine and the gas dynamics, the reader should consult related recent reports containing such information [17,5]. SEM imaging: In addition to the dark and bright field detection configurations, supplementary imaging was done in vacuum with the in-lens and ET detectors. Additionally, the specimen was mounted free of the previous shields but simply placed over the hole of a carbon Faraday cup for the purpose of observing only the surface image obtained in the standard “VP mode” of operation. Some of the details automatically recorded on micrographs should be cross-examined with the information provided in the legend of each figure. Thus, the detector recorded as “VPSE” on the micrographs is insufficient in differentiating the new imaging modes. The calibrated working distance is slightly different, i.e. as mentioned above at 2.8 mm from the recorded 3 mm, or otherwise stated in the legend of the figures. Some duplicate figures on the microgpraph and the legend are preserved should the demagnified printed micrographs produce too small print. All scanned images

Fig. 3. (a) Side view of the bright field assembly and (b) tilted side view of the dark field assembly corresponding to the diagrams in Fig. 2a and b.

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Fig. 4. VPSE images of the bright field (a) and of the dark field (b) configuration assemblies showing the copper grid specimen in focus, whilst the rim of the upper shield above and the holes on the corresponding Faraday cups below are out of focus.

have 1024  768 pixels and the recording time is provided separately with each image to give an idea of the relative amount of radiation dosage and imaging capability/efficiency of each detection mode. The test specimens are carbon nanotubes (CNTs) coated with PtCo particles prepared in the laboratory and resting on thin carbon films with nominal thickness in the range 5–15 nm as supplied by Agar for standard TEM work. From TEM micrographs, it appears that the particle sizes range between 3 and 6 nm. The specimens were prepared by dispersion in water and spread onto the carbon film covered copper grid (200 mesh). This type of specimen is thought to be a good choice for the current work, also because it has been used in other applications and makes a good reference material.

3. Results We started this investigation by initially placing the specimen grid with both sides exposed to free gaseous environment. This allows the electron beam to be transmitted both through the specimen and a considerable distance below it as in Fig. 1a. An image of carbon nanotubes coated with PtCo particles is shown in Fig. 1b, i.e. with a “bare” specimen where all signals contribute to the image. The increased noise required a much greater effort in focusing the beam often resulting in specimen damage by the

electron beam and raster shape formation on the image. All this prevented us from obtaining much higher magnification. The particles are hardly visible as bright spots, which means that the dark field electrons below together with contributions from BSE and SE above the specimen prevail over the canceling contrast of bright field (i.e. S (B) + S (C) > S (CX)). The latter condition is determined by the geometrical parameters of the given system, which may vary. This initial test by itself cannot inform us about the exact origin of contrast until further testing with appropriate controls is done. Fig. 5a is an image of same specimen taken with the system of Fig. 2a for bright field imaging. Contrast and resolution are greatly improved. The particles appear black with a gray level for the carbon nanotubes against a bright background field. Clearly, this has all the characteristics of a BF image, as expected. Due to a much improved signal-to-noise ratio (SNR), more time is available to obtain good images before the onset of visible radiation effects at relatively high magnifications. Fig. 5b is an image of CNTs taken with the system of Fig. 2b for dark field imaging. Indeed, as expected, we have a clear reversal of contrast with the particles appearing bright on gray carbon tubes against a dark field. Again, we have a dramatic improvement of contrast and resolution relative to the “bare” specimen imaging in Fig. 1b. Near the top of the image, the specimen appears suspended over a hole in the film producing a significant further improvement of contrast by eliminating the constant

Fig. 5. (a) Imaging according to Fig. 2a with PtCo particles on CNTs placed below the carbon film showing bright field contrast with good resolution at 49.34 kX, at 1.7 min. (b) Imaging according to Fig. 2b with PtCo particles on CNTs placed above the carbon film showing dark field contrast with good resolution at 66.94 kX magnification, at 1.3 min.

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Fig. 6. Bright field image at high magnification of 188.67 kX with evident carbon damage but still resolvable PtCo particles on CNTs placed on top of carbon film, at 25.4 s.

Fig. 8. Beam irradiation rasters and dark spots formed under high vacuum conditions during imaging with the in-lens detector at higher magnifications and switching back to the 23.52 kX recorded magnification.

(background) signal from the amorphous film. Carbon film and CNTs produce comparable signal intensity. Radiation indeed becomes eventually the limiting factor at the highest magnification as can be seen on a bright field image in Fig. 6, where at very high magnification the carbon nanotube appears to fade away, whilst the deposited particles are still clearly visible. This demonstrates that several nanometers are resolvable, i.e. a resolution is possible not far from the nominally specified instrument resolving power. The marked PtCo particle with 3.55 nm (like similarly visible particles) is consistent with particle size distribution between 3 and 6 nm, which are more clearly visible on micrographs obtained with conventional transmission electron microscopy in other work. It should be noted that the CNT specimen in this case is placed on top of the carbon film. The success of the above controls in signal separation indicates also the correct way for obtaining the “surface” information from thin specimens in an ESEM: It is achieved by simply placing the specimen over the hole of a carbon Faraday cup. This traps practically all transmitted signals (i.e. S (C) + S (CX)) leaving only the sum S (AB) + S (B ) alone, while it also limits the interaction volume only inside the thin specimen-plus-support-film, instead of extending it deeper in additional supporting substrate. Thus, Fig. 7a is an image taken with the VPSE detector followed for comparison by an image of the same field of view imaged with the available ET

detector in vacuum, as can be seen in Fig. 7b. Both images are comparable, whilst the first shows increased noise due to the S(AB) and associated beam loss that has already been demonstrated previously [17]. The important thing is that they are both also much better than the “bare” specimen in Fig. 1b, to which an uncontrolled multiplicity of signals is allowed to take part in the image formation. The VPSE image over the Faraday cup was also taken at longer working distance that better matched the fixed 25 Pa pressure of nitrogen. The amplification of GDD is a function of the product (pressure)  (distance) with an optimum depending on the parameters of the given instrument. Above all and throughout this work, the main problem arose from radiation effects becoming more intense and fast as we increased the magnification. To obtain the best output at the limit of the present configurations tested so far, the last resort was to focus over a small area and then immediately move to a neighboring virgin area to obtain a single image. It should be further noted that radiation effects are present both in high vacuum and in gas, even at lower accelerating voltage as can be seen by the rasters present in Fig. 8 that developed during prior imaging at higher magnifications with the in-lens detector in vacuum. The dark spots are caused by the stationary beam between scans. In other words, at first it seems that the electron beam and emanating signals are primarily responsible for

Fig. 7. (a) CNTs with PtCo particles placed over the hole of a carbon Faraday cup by use of the unmodified SUPRA VPSE detector, 58.03 kX, 30 kV, w_d ¼ 6 mm, at 2.7 min. (b) Same specimen and field of view as in previous but with ET electron detector in vacuum.

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the observed effects in both vacuum and gaseous conditions rather than the nitrogen ions formed around. However, an adsorbed surface nitrogen layer or outgassing gas from chamber walls may continue to take part in the same beam effects as we switch to vacuum operation. This and other interesting observations regarding separation of various types of signals like BSE, SE-II and others form a separate topic outside the purposes of this paper and may be reported in later works. Suffice it here to provide good evidence for the multipurpose potential of the available instrument. Interestingly, when using the BF and DF modes, there was no significant difference (observable or reproducible) in imaging the carbon nanotubes above or below the carbon film on which they were deposited, except in some special configurations other than those in Fig. 2a and b, where we noticed some deterioration of image quality. These special configurations may be again the subject of a continuing report in this area. Suffice it to say that we can expect a better image when the particles are placed above the holding film than below in view of the top–bottom effect [18]. However, this is observable only at resolutions near beam diameter, a limit not yet achieved at present due to specimen instability at higher magnifications as mentioned above.

4. Discussion It has now been demonstrated that ESEM can prove a much more powerful tool than just for specimen surface examination. It can be further used for the internal study of specimens. While this was already demonstrated by Bogner et al. [2] by introducing a conventional solid state BSE detector below the specimen, the use of the GDD approach can now lead to best possible outcomes in view of its unique properties. Transmission imaging has been done routinely practically at no cost with in-house made simple gadgets. The images presented were obtained easily and selected out of a much larger number stocked in a short period of time without any difficulty. The detection efficiency of GDD can, in principle, approach the 100% physical limit because the signal of interest is immersed in the detector and there can be practically zero signal loss not only by way of collection angle and particle number but also by way of total energy conversion to useful signal output. At present, it is only limited by simple engineering considerations in defining the collection (acceptance) solid angle for detection of a particular fraction of dark field signal, like low take-off angle or high take-off angle of DF electrons. The bright field collection angle can be even easier defined. In all cases, the total electron energy of a chosen signal fraction can be dissipated, converted and extracted directly in the gas, whereas the solid state BSE detector has often a threshold electron energy to overcome in the kV range, like a reported 13 kV threshold [20], before the electrons usefully dissipate their energy in the solid state material; although this passivation layer may be omitted, it offers protection to the detector. This is in addition to the requirement of using many different solid state pieces to cover the entire transmission solid angle and then process the electronic outputs. Another serious disadvantage of the solid state detectors is the radiation damage and contamination inflicted upon them by the energetic signal electrons, especially in the bright field configuration, where large amounts of beam energy is concentrated over the smallest possible volume of the solid state detector. GDD is completely free of any radiation and contamination damage to itself by its very gaseous nature. Such radiation damage may not be promptly mentioned by users and manufacturers alike but it must be diminishing the efficiency of the solid state detectors in proportion to the radiation dose, like it has been experienced with solid scintillating materials [19,12]. The

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GDD is clearly free of such damage, because the gas is continuously renewed and its particles do not undergo any permanent change in the detection process. Although the presented configurations have produced high resolution and contrast images in transmission mode, they still lie far from optimum conditions. Both S(B) (for SEM work) and S(C) with S(CX) (for TESEM work) reaching the PMT in the present experiment are generally less and often much less than the corresponding total signals exiting the specimen in addition to being mixed with white background noise by the incident electron beam. To remedy the losses and improve contrast and resolution in this regard, it is planned to apply the following suggested improvements: For BF imaging, the collection angle should not exceed the angle needed to collect only the electrons pertaining to this mode, while the travel distance CD per Fig. 2a becomes shortest to minimize electron losses by gaseous scattering. The choice of detector aperture should be subject to further testing in view of the conflicting requirements for making the electron probe aperture equal to the detector aperture (per conventional STEM) but the detector aperture large enough to contain the scanned raster at a particular magnification. Thus, in practice the detector aperture is larger than the probe aperture and it inevitably contains some dark field electrons. For DF imaging the collection angle should be defined so as to minimize interference by bright field electrons, while it allows maximum escape route in the gas (angle and distance) for the DF electrons. This again necessitates the travel distance CD per Fig. 2b to be as short as possible, which, for example, can be achieved with a needle size trap for the BF electrons [15]. By such means, we may reconcile the requirement to keep the shortest distance CD to minimize interference of BF electrons scattered by gas and smallest (i.e. optimum) detector angle to free a maximum number of DF electrons to travel a maximum distance in the gas. The generally small detector apertures necessitate means to allow specimen movement in the direction normal to the beam independently from the fixed placement of aperture relative to the specimen grid per Fig. 4a and b. By such means, the entire grid can be surveyed instead of only the limited field of view imposed by the detector aperture in both BF and DF modes. This is a matter of technological refinement in specimen/aperture handling, which will provide a solution towards reaching the optimum imaging conditions outlined above. A large improvement can be further achieved by eliminating the unnecessary primary electron beam losses in the initial environmental distance e_d imposed by the current machine [17]. It is planned to eliminate the excess (difference) travel distance e_d  w_d as per Fig. 1a in forthcoming work. The photon collection efficiency by the existing PMT is thought to be also away from optimum, which points to another significant improvement. Conversion of electron signal to photons may not be limited only in the gas, but the surfaces upon which both DF and BF terminate can act as electron to photon converters in an equivalent fashion done by Crawford and Liley [4], who converted the transmitted fast electrons to SE for detection by the ET detector in a conventional SEM. The GDD sensitivity also depends on pressure, nature of gas, and proportional amplifier bias applied on the corresponding electrode. For example, a particular improvement can be achieved by “seeding” the gas with special gas admixtures to significantly improve signal conversion to photons/electrons and eventual detection [9]. By any or all of the above and other improvements, it is expected to greatly improve upon the present already good results. This will further aid the use of higher chamber pressure and lower

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accelerating voltage for the examination of wet specimens and low bearing contrast biological and living material, such as already achieved by Bogner et al. [2], but hopefully with even higher contrast and resolution. We are currently limited only by the artificial limitations imposed by the specifications of this particular model of microscope, not by any physical limits which are yet to be reached. This limit should be a resolution at beam spot diameter, which should be same both in vacuum and in gas according to a fundamental theorem of ESEM [8]. The same detection configurations disclosed here can be adapted to other models of same base microscopes already specified to operate at higher pressure (i.e. by Zeiss company). Similarly, the same detection configurations can be adapted for use in the other class of ESEM machines that are based on the ionizationGDD (e.g. by FEI Company). The difference will be in the use of equivalent electrostatic shields and (mechanical) traps of the relevant signals, since the photons are not incorporated in their detection system. In this regard, systems of electrodes and grids suitably biased will serve to separate the various detection and amplification volumes in the specimen chamber as described in detail in the original theory of the gaseous detection device [9]. There are some advantages and disadvantages between scintillation and ionization GDD as, for example, the scintillation readily allows examination at true TV scanning rates [10]. If we are to take full advantage of all these possibilities and improvements in signal detection, of no less importance is the handling of specimens and gas pressure controls available: For example, it has been shown that it is possible to insert and maintain a specimen fully wet ready for examination at room temperature in a very short time of less than 60 s [8, pp. 238–240], whereby cooling of the specimen has become redundant [16]. Notwithstanding all these prior and relatively old achievements, biological specimens in some modern machines still require about 15 min [24] by the Cameron and Donald [3] method that was thought necessary to counter the harsh pumping regime used by these machines. As a result, we often see reports on collapsed bacteria due to dehydration, whereas the ultimate limitation is expected to come only from radiation damage at the highest possible magnifications. Pending all these improvements, namely, the operation of an ESEM and TESEM close to their physical limits, we still hope to be able to observe the movement of living cells. The overall concepts of ESTEM and TESEM discussed herewith have already been disclosed in patent application forms in the description of a universal electron scanning microscope (UESM) [15], which allows for the examination of specimens at much higher pressure (i.e. up to atmospheric) and at much higher accelerating voltages as typically employed in conventional STEM. However, the present work is a precursor demonstration for the first time in practice of those ideas and disclosures. Finally, the present work has demonstrated a significant step towards unification of all main modes of electron microscopy in one instrument: Based on previous works and the present report, we have shown high resolution SE images in vacuum, natural surface images of bulk specimens in a gaseous environment and both modes of DF and BF in transmission environmental scanning electron microscopy. Wet specimens are already a fact in TESEM and, hence, all evidence is now provided for the manufacture of a universal electron scanning microscope for general use.

5. Conclusion An ESEM can operate also as a TESEM with minimal modifications. In particular, those instruments that employ the scintillation-GDD are practically ready to yield both bright and dark field imaging equivalent to conventional STEM almost at no cost.

They may yield unique information on account of the low accelerating voltage contrast, such as in biological materials. Specimen holders with appropriate shields or traps can separate the various fractions of signals, not only in transmission mode but also in bulk (SEM) mode to improve contrast and select particular information. Bright field images of high resolution have been produced by shielding the photons and electrons above and below the specimen section selectively, i.e. except for the unscattered primary beam that is separated out through a small aperture below the specimen and is allowed to dissipate its energy in the environmental gas producing photons. The latter are detected by the PMT present to record a bright field image in the usual way. Similarly, dark field images of high resolution have been produced by eliminating the transmitted beam and all other signals except for the dark field electrons that are allowed to dissipate their energy in the gas and produce photons, and so on. Beyond the proof of principle of a TESEM with a scintillationGDD, there is good purpose for significant improvements in all types of ESEM (including ionization-GDD) by allowing them to operate as TESEMs and producing high contrast and resolution with the available powerful modern instruments and best electron optics.

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