MICROSCOPY RESEARCH AND TECHNIQUE 73:225–228 (2010)
Influence of Acceleration Voltage on Scanning Electron Microscopy of Human Blood Platelets E. PRETORIUS* Faculty of Health Sciences, Department of Anatomy, School of Medicine, University of Pretoria, South Africa
KEY WORDS
platelets; scanning electron microscopy; acceleration voltage
ABSTRACT Scanning electron microscopy (SEM) is used to view a variety of surface structures, molecules, or nanoparticles of different materials, ranging from metals, dental and medical instruments, and chemistry (e.g. polymer analysis) to biological material. Traditionally, the operating conditions of the SEM are very important in the material sciences, particularly the acceleration voltage. However, in biological sciences, it is not typically seen as an important parameter. Acceleration voltage allows electrons to penetrate the sample; thus, the higher the acceleration voltage the more penetration into the sample will occur. As a result, ultrastructural information from deeper layers will interfere with the actual surface morphology that is seen. Therefore, ultimately, if acceleration voltage is lower, a better quality of the surface molecules and structures will be produced. However, in biological sciences, this is an area that is not well-documented. Typically, acceleration voltages of between 5 and 20 kV are used. This manuscript investigates the influence of acceleration voltages ranging from 5 kV to as low as 300 V, by studying surface ultrastructure of a human platelet aggregate. It is concluded that, especially at higher magnifications, much more surface detail is visible in biological samples when using an acceleration voltage between 2 kV and 300 V. Microsc. Res. Tech. 73:225–228, 2010. V 2010 Wiley-Liss, Inc. C
INTRODUCTION Scanning electron microscopy (SEM) is used to view a variety of surface structures, molecules, or nanoparticles of different materials, ranging from metals, dental and medical instruments, and chemistry (e.g. polymer analysis) to biological material. Traditionally, the operating conditions of the SEM are very important in the material sciences. Particularly, because SEM is often used to evaluate surface contamination and machining defects in dental and other medical instruments. Knowledge of the operating conditions of the SEM, in particular, the accelerating voltage is essential to properly interpret images of such material (Stowe et al., 2004). The SEM irradiates an area with a finely focused electron beam that penetrates the specimen. The penetration of the primary electrons in the solid (interaction volume) depends on its average atomic number, the beam energy, and specimen tilt (Phillips et al., 2003). Accelerating voltage is the voltage applied to the filament. Together with the application of a small current, it will cause the electrons to leave the filament and penetrate the material. With an increase in the acceleration voltage, more penetration into the sample will occur. The accelerating voltages often used in SEM image analysis of biological material vary between 5 and 20 kV. However, most samples can achieve various benefits from the combination of low voltage and low vacuum (Oho et al., 2000). With a low-accelerating voltage, the beam penetration is small and therefore more information regarding the surface of the structure is obtained. However, when using a high-accelerating voltage, more internal structure information is C V
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obtained, and therefore fine details on the surface will be lost. Literature suggests that in fields like polymer science and engineering, accelerating voltage plays a crucial role. It seems that a lower voltage produces images that show much more detail. Stowe and coworkers (2004) demonstrated the importance of using low-accelerating voltages to detect surface features including contamination on NiTi rotary and hand files and conclude that even recent studies may have significantly underestimated the amount of nonmetallic debris (from the manufacturing process or from biological contamination) present on the surface of such instruments. In polymer science, SEM images are used to digitally analyze particle size and dispersion (Yan˜ez and Barbosa, 2003). It is now believed that kilovolt has an important influence on area measurement in SEM images (Yan˜ez and Barbosa, 2003). In a study by Yan˜ez and Barbosa (2003), the authors found that the higher the accelerating voltages, the greater the error at high magnification for polymer samples. As the beam energy increases, the primary electrons penetrate more deeply into the solid specimen, producing lowresolution signals. These signals degrade the image and surface details, which became less well defined. The authors concluded that images of polymer samples *Correspondence to: E Pretorius, BMW Building, PO Box 2034, Faculty of Health Sciences, University of Pretoria, Pretoria 0001, South Africa. E-mail:
[email protected] Received 18 June 2009; accepted in revised form 5 August 2009 DOI 10.1002/jemt.20778 Published online 19 January 2010 in Wiley InterScience (www.interscience. wiley.com).
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must be taken at lower accelerating voltages so that the desired surface details can be imaged clearly. Interestingly, in analysis of biological material, the accelerating voltage is rarely mentioned in publications. In the1980s and1990s, authors like Pawley, Erlandsen, and Joy published extensively on low-voltage high-resolution SEM of biological samples (Joy and Pawley, 1992; Pawley and Erlandsen, 1989; Pawley, 1997). Pawley and Erlandsen (1989) mentioned that dried biological samples are low in scattering power, nonconducting, and sensitive to radiation damage and that these facts complicate the choice of the optimum beam voltage Vo at which they should be observed in the SEM, because they add as variables the type and thickness of the coating material and degradation/contamination of the specimen by the beam. The authors also mentioned that high-resolution SEM could typically only be carried out at relatively high Vo (20–30 kV), because available equipment could not produce small beam diameters at low Vo. Ushiki and coworkers (1998) pointed out that a better resolution surface image of the noncoated biological specimens were obtained at an accelerating voltage of 5 kV, whereas the images at 10–20 kV contained information beneath the surface of the specimens. They also mentioned that a low (3–5 kV) accelerating voltages is useful and should rather be used for the three-dimensional analysis of the surface morphology of biological noncoated samples. Hashizume and coworkers (1997) used backscattered electron (BSE) imaging SEM to a study on the mineral density of the bone surface. The neonatal and adult mouse parietal bones freed of the periosteum, and covering cells were examined in a field emission scanning electron microscope equipped with a high-sensitivity BSE detector at 1–30 kV accelerating voltages. The authors suggested that low-accelerating voltage SEM using BSE provides new information on the distribution of the osteoid and also the bone matrix calcification under both normal and pathological conditions. However, even now, nearly 20 years later, acceleration voltage in biological material is typically set at 5 kV and sometimes even higher—up to 20 kV, and conversely, rarely or never queried reviewers of manuscripts. Currently, new SEM technology allows the biological research field to use even lower acceleration voltages than 1 kV. The question that now arises is whether Biologists have been missing surface morphology that might provide us with better results, because we have not played around with this setting or have not had access to equipment that can accommodate very lowkilovolt settings. Surface ultrastructure can play an important role as marker for diseases, including HIV/AIDS, cancer, and conditions like thrombocytopenia. Structures like platelets and fibrin networks may provide important diagnostic information to clinicians. It is in this field of Biology where low-kilovolt settings may be of particular importance. The current research therefore studies a human platelet aggregate at different acceleration voltages and argues for the use of lower acceleration voltage analysis in biological ultrastructural analysis. MATERIALS AND METHODS Equipment Used A Zeiss ULTRA plus FEG-SEM with InLens capabilities, using nitrogen gas and ultra high-resolution BSE
imaging was used to study surface morphology of a human platelet aggregate. This instrument can be applied in Material Science, Life Science, and the Semiconductor analysis. Micrographs were taken at 5 kV, 2 kV as well as 300 V. This instrument is located in the Microscopy and Microanalysis Unit of the University of Pretoria, Pretoria, South Africa. Preparation of Fibrin Clots Fresh platelet-rich plasma from a healthy donor was prepared by drawing 40 mL of blood. Blood was centrifuged at 1,000 rpm (maximum RCF 5 17.523g; 1,250 g) for 2 min. Human thrombin (provided by the South African National Blood Service) was used to prepare these fibrin clots from the donor. The thrombin solution is at a concentration of 20 U/mL and is made up in a biological buffer containing 0.2% human serum albumin. When thrombin is added to platelet-rich plasma, fibrinogen is converted to fibrin and intracellular platelet components, for example, transforming growth factor, platelet-derived growth factor, and fibroblastic growth factor are released into the coagulum. Twenty microliters of the PRP was mixed with 20 lL of human thrombin on a 0.2-lm millipore membrane to form the coagulum (fibrin clot). This millipore membrane was then placed in a Petri dish on filter paper dampened with phosphate buffered saline (PBS) to create a humid environment and placed at 378C for 10 min. This was followed by a washing process where the millipore membranes with the coagula were placed in PBS and magnetically stirred for varying times (45, 90, and 120 min). This was done to remove any blood proteins trapped within the fibrin network (Pretorius et al., 2007, 2008). Preparation of Washed Fibrin Clot for SEM Washed fibrin clots were fixed in 2.5% glutaraldehyde in Dulbecco’s phosphate buffered saline (DPBS) buffer with a pH of 7.4 for 1 h. Each fibrin clot was rinsed thrice in phosphate buffer for 5 min before being fixed for 1 h with 1% osmium tetraoxide (OsO4). The samples were rinsed thrice with distilled water for 5 min and dehydrated serially in 30, 50, 70, 90, and three times with 100% ethanol. The SEM procedures were completed by critical point drying of the material, mounting, and examining the tissue with a ZEISS ULTRA Plus FEG-SEM. RESULTS AND DISCUSSION Figure 1 shows SEM micrographs of a single-platelet aggregate at 20,0003 magnification on machine (scale bar 5 1 lm). Figure 1a the SEM is set at 5 kV, Figure 1b is set at 2 kV, and Figure 1c is set at 300 V. Figure 2 shows SEM micrographs of the single platelet aggregate at 50,0003 magnification on machine (scale bar 5 200 nm). Figure 2a the SEM is set at 5 kV, Figure 2b is set at 2 kV, and Figure 2c is set at 300 V. Figure 3 shows SEM micrographs of the single-platelet aggregate at 100,0003 magnification on machine (scale bar 5 100 nm). Figure 3a the SEM is set at 5 kV, Figure 3b it is set at 2 kV, and Figure 3c is set at 300 V. At 5 kV, the beam penetrates deeper into the material and therefore, more information from the lower/ deeper layers interferes with the view of the tissue. Microscopy Research and Technique
INFLUENCE OF ACCELERATION VOLTAGE ON PLATELET MORPHOLOGY
Fig. 1. a: SEM micrograph of 5 kV (20,0003 magnification on machine). Scale bar 5 1 lm. b: SEM micrograph of 2 kV (20,0003 magnification on machine). Scale bar 5 1 lm. c: SEM micrograph of 300 V (20,0003 magnification on machine). Scale bar 5 1 lm.
Therefore, at lower kilovolt, particularly at 300 V, more surface structure information will be seen. Figures 1a–1c show the platelet aggregate at low magnification (20,0003 magnification on machine). At low magnification, the micrograph at 2 kV and 300 V, much more surface structure is seen (note details of Microscopy Research and Technique
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Fig. 2. a: SEM micrograph of 5 kV (50,0003 magnification on machine). Scale bar 5 200 nm. b: SEM micrograph of 2 kV (50,0003 magnification on machine). Scale bar 5 200 nm. c: SEM Micrograph of 300 V (50,0003 magnification on machine). Scale bar 5 200 nm.
ultrastructure indicated with arrows) than the micrograph taken at 5 kV. This is also seen with 50,0003 magnification (Figs. 2a–2c) and 100,0003 magnification (Figs. 3a–3c). With higher magnification, more surface structure detail is seen when using low-accelerating voltage (2 kV and 300 V) compared to the 5 kV
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more defined (Figs. 3b and 3c, black arrows), and bulbous areas appear more pronounced with more definition (Figs. 3b and 3c, black lines indicating bulbous area on platelet). Where previous research suggested studying surface detail in material science at a low-acceleration voltage, this study confirms it an absolute necessity. It is therefore suggested that in biological material, especially when there is an interest in determining changes at a molecular morphology level, we should be using accelerating voltages of in the range of 2 kV. However, acceleration voltage is not the only parameter that could play a role in viewing surface ultrastructure. Specimen preparation is surely also one of the important parameters that play a role in the quality of the image. In future studies all the different parameters should be investigated to ultimately produce a micrograph that represents the true morphology of the structure.
ACKNOWLEDGMENTS I acknowledge A. Botha for technical help using the SEM as well as HM Oberholzer for technical assistance with preparing the biological samples.
REFERENCES
Fig. 3. a: SEM Micrograph of 5 kV (100,0003 magnification on machine). Scale bar 5 100 nm. b: SEM micrograph of 2 kV (100,0003 magnification on machine). Scale bar 5 100 nm. c: SEM micrograph of 300 V (100,0003 magnification on machine). Scale bar 5 100 nm.
micrograph, which looks hazy, with little definition visible. However, at 100,0003 magnification, very little differences between 2 kV and 300 V are seen. Differences are mainly seen at crevices, where they are perhaps
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