Human Bloodstains on Biological Materials: High-Vacuum Scanning Electron Microscope Examination Using Specimens without Previous Preparation

2013 ◽  
Vol 19 (2) ◽  
pp. 415-419 ◽  
Author(s):  
Policarp Hortolà

AbstractStudies of human bloodstains on nonbiological materials have been previously carried out using a high-vacuum scanning electron microscope (HV-SEM) in secondary-electron mode without any sample treatment. To assess whether biological substrates can affect the morphology of human erythrocytes in bloodstains, three fragments of different biological material (bone, shell, and wood) were smeared with peripheral human blood. Afterward, the bloodstains were directly examined in secondary-electron mode by an HV-SEM following a procedure initially standardized to be used in uncoated human bloodstains on stone. The obtained results suggest that HV-SEM is suitable for examining untreated bloodstains on biological substrate and that the morphology of erythrocytes in human bloodstains is not affected by the biological nature of the substrate. A cautionary issue regarding bloodstains on nondehydrated biological substrates is that the waiting time required for initiating the HV-SEM examination is by far higher than when using inorganic bloodstain substrates.

Author(s):  
James B. Pawley

Used in the secondary electron mode, the Scanning Electron Microscope (SEM) produces an image of the outside surface of a microscopic sample which looks very similar to what one might expect to see if the sample was a diffusely illuminated macroscopic object viewed with the unaided eye. Part of the familiarity of such an image is associated with the fact that one seems to look at the sample rather than through it, as in the case with the conventional electron microscope or the high resolution light microscope. A resulting limitation is the fact that an object of interest cannot be observed if it is below the outer surface. It has been shown (Gane and Bowden 1968) that useful surface hardness information can be obtained on a micro scale by observing the deformation produced when a small stylus, attached to a D'Arsonval meter movement, is brought to bear on the surface of a sample while it is in the SEM.


Author(s):  
L.W. McDonald ◽  
R.F.W. Pease ◽  
T.L. Hayes

In previous studies from this laboratory the scanning electron microscope has been used to examine biological materials in the cathodo-luminescense and secondary electron modes. In these studies intact cells or even entire insects have been examined, some in the living state. Epithelial surfaces have been exposed and examined. Prior to the work to be described, no reports of the examination of tissue sections in the scanning electron microscope have been found, although sufaces of solid one millimeter cubes of tissue have been examined.In the present work blocks of solid tissue fixed in buffered aldehyde have been dehydrated in graded alcohols, embedded in paraffin, section at 4μ and examined successfully in the scanning electron microscope. These sections have been over 1 cm square and have been stained for subsequent comparative examination with the light microscope. With the scanning electron microscope in the secondary electron mode, magnifications of X5,000 have been found useful. In addition to the increased resolution as compared to the light microscope, a three dimensional image is obtained. An advantage over the conventional electron microscope is that tissue areas 1,000 times greater may be examined in the large sections without any obscuring grid bars, again with the three dimensional image. With the cathode ray display tube used, magnifications range from X30 to over X20,000


Author(s):  
W. R. Bottoms

The vacuum system of any electron optical instrument effects the contamination rate, electron source life, the quality of the electron source which can be employed, vibration amplitudes and stray magnetic field levels. It is particularly important for the scanning electron microscope where the object of primary interest is a specimen surface which can be altered by contamination. If we extend our investigations to employ Auger electron spectroscopy for surface chemical analysis, the requirements on the vacuum system are much more stringent. It is necessary that the chemical composition of the surface monolayer is not appreciably altered during the time required to take Auger spectra. The vacuum level required to accomplish this is dependent on the specimen material and the chemical composition of the ambient gas.Commercially available equipment can be modified to provide a vacuum environment maximizing the analytical capabilities of the instrument. The gas loads from the specimen and electron gun chambers of the instrument are minimized by utilizing only materials with favorable outgassing rates, and employing a gentle bakeout to remove water and other loosely bound gases on the system surfaces.


Author(s):  
Oliver C. Wells

The low-loss electron (LLE) image in the scanning electron microscope (SEM) is useful for the study of uncoated photoresist and some other poorly conducting specimens because it is less sensitive to specimen charging than is the secondary electron (SE) image. A second advantage can arise from a significant reduction in the width of the “penetration fringe” close to a sharp edge. Although both of these problems can also be solved by operating with a beam energy of about 1 keV, the LLE image has the advantage that it permits the use of a higher beam energy and therefore (for a given SEM) a smaller beam diameter. It is an additional attraction of the LLE image that it can be obtained simultaneously with the SE image, and this gives additional information in many cases. This paper shows the reduction in penetration effects given by the use of the LLE image.


Author(s):  
M.G. Rosenfield

Minimum feature sizes in experimental integrated circuits are approaching 0.5 μm and below. During the fabrication process it is usually necessary to be able to non-destructively measure the critical dimensions in resist and after the various process steps. This can be accomplished using the low voltage SEM. Submicron linewidth measurement is typically done by manually measuring the SEM micrographs. Since it is desirable to make as many measurements as possible in the shortest period of time, it is important that this technique be automated.Linewidth measurement using the scanning electron microscope is not well understood. The basic intent is to measure the size of a structure from the secondary electron signal generated by that structure. Thus, it is important to understand how the actual dimension of the line being measured relates to the secondary electron signal. Since different features generate different signals, the same method of relating linewidth to signal cannot be used. For example, the peak to peak method may be used to accurately measure the linewidth of an isolated resist line; but, a threshold technique may be required for an isolated space in resist.


Author(s):  
G.D. Danilatos

The environmental scanning electron microscope (ESEM) has evolved as the natural extension of the scanning electron microscope (SEM), both historically and technologically. ESEM allows the introduction of a gaseous environment in the specimen chamber, whereas SEM operates in vacuum. One of the detection systems in ESEM, namely, the gaseous detection device (GDD) is based on the presence of gas as a detection medium. This might be interpreted as a necessary condition for the ESEM to remain operational and, hence, one might have to change instruments for operation at low or high vacuum. Initially, we may maintain the presence of a conventional secondary electron (E-T) detector in a "stand-by" position to switch on when the vacuum becomes satisfactory for its operation. However, the "rough" or "low vacuum" range of pressure may still be considered as inaccessible by both the GDD and the E-T detector, because the former has presumably very small gain and the latter still breaks down.


Author(s):  
R. B. Neder ◽  
M. Burghammer ◽  
Th. Grasl ◽  
H. Schulz

AbstractWe developed a new micro manipulator for mounting individual sub-micrometer sized single crystals within a scanning electron microscope. The translations are realized via a commercially available piezomicroscope, adapted for high vacuum usage and realize nanometer resolution. With this novel instrument it is routinely possible to mount individual single crystals with sizes down to 0.1


2008 ◽  
Vol 16 (4) ◽  
pp. 62-63
Author(s):  
V.M. Dusevich ◽  
J.H. Purk ◽  
J.D. Eick

Coloring pictures is an educational exercise, which is fun, and helps develop important skills. Coloring SEM micrographs is especially suitable for electron microscopists. Color micrographs are not just great looking on a lab wall; they inspire both microscopists and students to exercise digital picture manipulation. Many microscopists enjoyed looking at the beautiful color micrographs by D. Scharf, but were frustrated to learn they needed a very particular scanning electron microscope equipped with multiple secondary electron detectors in order to color their own pictures. Fortunately, there are other ways to color SEM micrographs. Most SEMs are equipped with at least two detectors, for secondary and backscattered electrons.


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