Operation and Imaging

2.2.1. The Chamber Gas

VPSEM and ESEM operate with elevated chamber gas pressures. A range of gases can be used, and the attributes of the different gases have been extensively

Scanning Electron Microscope Chamber
Fig. 1. A schematic variable pressure scanning electron microscope (VPSEM) or environmental SEM (ESEM) column illustrating the role of the pressure-limiting apertures (PLAs 1-3) and the differential pumping of the column. Some VPSEM may only have 1 or 2 PLAs.

investigated (8). For imaging of biological samples two gases, air and water vapor, dominate current usage. Water vapor is without question the most effective gas, and the commercial ESEM range has used it since the original development. Water vapor has ideal ionization characteristics and so it behaves well as the imaging gas. Second, a major benefit of VPSEM and particularly ESEM is the ability to image hydrated samples and this requires water vapor as the chamber gas. In VPSEM, the rate of dehydration of biological samples (see Section 3) is obviously reduced significantly if water vapor is used.

Water vapor can be used in any VPSEM by the following simple modification, BUT only with support of the appropriately trained and suitably responsi ble service personnel. A vacuum flask, with protective wrap, can be attached to the air inlet used for supplying the chamber gas. The flask should be partially filled with DI water. Never fill the flask to more than 40% capacity because the space above the liquid is the vacuum reservoir. Overfilling will resulting in "boiling" of the water as the result of rapid pressure reduction over the liquid surface. The compatibility of the pumping system MUST also be checked prior to this conversion.

2.2.2. Gas-Electron Interactions

The basic electron-gas interactions that occur in VPSEM are reasonably well understood, and the physics is well defined (9). The main practical effect is a reduction in the image signal-to-noise (SNR) occurs because of the loss of scattered electrons out of the primary beam probe as the chamber gas pressure is increased (see Fig. 2). These scattered primary electrons generate a signal from the sample that is a noise component as it lacks spatial coherence. The scattered electrons are a more significant factor when performing X-ray microanalysis as the X-rays generated by them contribute spurious data from outside of the primary analysis area.

1. Gas path length = distance from final aperture to sample surface (this is often NOT the same as working distance because in many instruments the final aperture projects into the sample chamber below the plane of the objective lens).

2. Chamber gas pressure.

3. Chamber gas characteristics.

4. Primary beam energy (accelerating voltage).

2.2.3. Secondary Electron Imaging

Conventional Everhard-Thornley and the newer in-lens SE detectors do not operate under elevated gas pressure conditions because a high-voltage discharge will occur. A range of secondary electron detectors have been developed based on the attraction of the secondary electrons to a biased element. In all cases, the emitted electrons interact with the chamber gas and a cascade amplification of the electron signal occurs toward the biased element. The emitted electrons accelerate to a point where they ionize a gas molecule, with the resultant electrons then repeating the process. The resultant electron signal is termed the gaseous secondary electron (GSE) signal. The most common GSE detector measures the signal flux in the biased element (2); a second variant measures the specimen current that is in fact the ion flux, arising from the electron-gas amplification, flooding the sample surface (5). The third common GSE detector variant measures the luminescence from the gas as it recombines (10). The basic electron gas cascade amplification process and the various GSED detector arrangements are shown in Fig. 3.

Fig. 2. Modeled effects of scattering a 10-kV primary electron beam by water vapor with a 6-mm working distance (= gas path length) and for chamber gas pressures of (A) 27 Pa (0.2 torr), (B) 133 Pa (1.0 torr), (C) 565 Pa (4.0 torr), and (D) 1120 Pa (8 torr). The values in each pangel represent the diameter containing 90% of primary beam electrons on the sample surface. Bar = 1 mm.

Fig. 2. Modeled effects of scattering a 10-kV primary electron beam by water vapor with a 6-mm working distance (= gas path length) and for chamber gas pressures of (A) 27 Pa (0.2 torr), (B) 133 Pa (1.0 torr), (C) 565 Pa (4.0 torr), and (D) 1120 Pa (8 torr). The values in each pangel represent the diameter containing 90% of primary beam electrons on the sample surface. Bar = 1 mm.

Pressure Electron Microscope
Fig. 3. A schematic illustrating (A) the gas amplification process and (B) the gaseous secondary electron (GSE) detector variants available for variable pressure scanning electron microscopy and environmental scanning electron microscopy.

Primary beam and backscattered electrons contribute to this detected GSED signal. The level of contribution is a function of the scattering parameters (see Subheading 2.2.2.), and among those parameters, scattering has a strong inverse relationship to the primary beam energy, i.e., at low beam energies very high degrees of scattering are encountered. Some detector variants collect in parallel a backscattered electron signal and subtract it from the GSE signal to provide a more pure SE type signal. Comprehensive fundamental accounts of the gas amplification processes and contrast mechanisms have been presented (11).

All GSED depend upon the bias voltage on the positive detector element to generate the necessary gas amplification process. The bias voltage is commonly labeled "contrast" on the GSED controls. Practically, it is useful to note the value when good imaging conditions are present to allow consistency and reproducibility of imaging. The upper level of the GSED bias is normally limited by the value where an electrical discharge between the detector and the sample, or arcing, occurs rather than the operational range of the electronics. The specific voltage depends on gas pressure, type, detector design, and sample/stage configurations, i.e., the general operating conditions. The usual practice is to maximize the GSED bias to just below the discharge condition to provide the best image SNR ratio.

2.2.4. Sample Charge Cancellation

Positive ions are generated by the various electron-gas interactions in the sample chamber. The earliest and simplest VPSEM operated with low Ar gas pressures (typically 10-15 Pa) introduced in the chamber just above the sample. This arrangement produced sufficient positive ions to cancel charge and so to allow the BSE imaging (BSEI) and X-ray microanalysis of the uncoated and non-conductive mineral samples under study (1).

The gas amplification present with GSED operation produces a positive ion flood that far exceeds the primary electron beam current. The ions move slowly relative to the electrons, which can be observed practically as a brief instability of the SE image when moving to a new area on the sample, depending on sample and conditions. Where ions dominate the signal, a smearing of the image is present at faster beam scan rates. Most importantly, the ions can recombine and thereby reduce the GSE signal. A second detector element, grounded or slightly negatively biased, introduced between sample and biased GSE detector element will reduce this effect and can markedly improve image quality under some conditions (12,13).

2.2.5. BSE and Cathodoluminescence (CL) Imaging

The original imaging mode in VPSEM was based on both solid state and scintillator based BSED and excellent results remain obtainable on biological

Bse Imaging Conditions
Fig. 4. A mixed GSE-BSE image of a biological sample (cancer cell grown on glass) at low magnification in the VPSEM compared with the same sample coated and imaged by conventional SE under high vacuum conditions (Courtesy of Zeiss SMT Pty Ltd.)

samples (see Fig. 4). The practical limitation for both VPSEM and ESEM is in the BSED assembly thickness as these detectors are normally positioned directly above the sample (see Fig. 5). A thick BSED assembly will severely compromise the minimum achievable working distance and should be avoided. Practically, the consequent long gas path length will limit the achievable image quality at moderate chamber gas pressures and/or low accelerating voltages.

Panchromatic CL imaging is routinely achievable with conventional CL detectors. The only compromise is that the emissions from gas luminescence and charge contrast imaging (see Subheading 3.4.) can interfere with the CL signal. Practically the contributions to CL from these other processes can be easily determined by imaging with the GSED bias on and then to identify the various signal contributions. Fiberoptic-based spectral CL is also effective in VPSEM (14). There has only been limited application of CL in the study of biological samples to date but the technique is relevant to investigating bioluminescence

Fig. 5. A CCD camera view of a solid state backscattered electron detector (BSED) in place above the sample with an off-axis luminescence-based gsed. The narrow clearance between sample and detector and the increased gas path length due to the presence of the BSED are evident. GSED, gaseous secondary electron detector.

Fig. 5. A CCD camera view of a solid state backscattered electron detector (BSED) in place above the sample with an off-axis luminescence-based gsed. The narrow clearance between sample and detector and the increased gas path length due to the presence of the BSED are evident. GSED, gaseous secondary electron detector.

and similar processes. The new identification of a charge contrast effect in coral and other tissues (see Subheading 3.4.) will increase interest in this area.

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