Dual beam instruments provide high resolution and analysis in three dimensions for Brasília’s Instituto Nacional de Criminalistica.

Microscopy has long been associated with forensic science. Sherlock Holmes’ magnifying glass is iconic of the field of criminal investigation. The association continues in popular media today. Who has not seen the graphic sequences, zooming to high magnification, sprinkled liberally through most episodes of CSI? Forensic science and microscopy have come a long way since the days of Sherlock Holmes. High-powered scanning electron microscopes (SEM) can now reveal details down to the nanometer scale, one billionth of a meter. Investigators at Brasília’s Instituto Nacional de Criminalistica (INC/National Criminalistic Institute), the central crime lab for Brazil’s Federal Police Department, recently added a state of the art dual beam system to their laboratory. Dual beam instruments combine the high resolution imaging capability of SEM with the cross sectioning and sample manipulation capabilities of a focused ion beam (FIB). In addition, the instrument acquired by the INC (Quanta 200 3D DualBeam, FEI) includes specialized “environmental” (ESEM) capabilities that help to preserve the evidentiary integrity of the sample.

SEMs offer much higher resolution than optical microscopes, which are limited by the wavelength of the light they use to resolutions of a few tenths of a micrometer. SEMs form an image of the sample by scanning a finely focused electron beam over the surface and measuring the intensity of various signals caused by interactions between the beam and the sample at each point in the scanning pattern. These measurements are then mapped as brightness levels in a grayscale image. The resolution of the image is determined by the minimum size of the spot into which the beam can be focused on the sample surface and the size of the volume from which signals emanate as the beam electrons penetrate and scatter within the sample. Most SEMs can resolve features down to a few nanometers, 100 times better than optical microscopes. In terms of useful magnification, optical microscopy is useful up to about 1,000X and SEM up to 100,000X or more.

In addition, SEM offers much greater depth of field than optical microscopy. The high numerical apertures required for high magnification and resolution in an optical microscope limit the thickness of the region that is in focus. In contrast, the narrow convergence angle of the electron beam permits very deep focal fields and three dimensional images. Dual beam instruments add to this three dimensional capability by allowing the investigator to use the focused ion beam to cut precisely controlled cross sections into the sample to reveal subsurface detail. The two beams are configured so that the electron beam “looks at” the surface cut by the ion beam.

SEM also offers the ability to determine the sample’s elemental composition on a microscopic scale. As the electron beam interacts with sample atoms they generate X-rays. The energy of each X-ray is characteristic of the type of atom from which it originates. By counting the X-rays and measuring their energy, it is possible to determine the type and relative abundance of atoms within the volume of interaction corresponding to the position of the beam. Because X-rays can travel relatively long distances through the sample (compared to the signals used for imaging), the spatial resolution of the analysis is typically on the order of a few micrometers. The energies or wavelengths of the X-rays are measured using energy dispersive or wavelength dispersive spectrometers (EDS or WDS).

Because electrons in the beam are easily scattered by gas molecules, conventional SEMs must operate with high vacuum in the electron column, where the beam is formed, and the sample chamber. This requires that the sample be compatible with a vacuum, i.e. dry, clean and not outgassing. If it is not conductive it must be coated with a conductive material to allow it to dissipate the charging effects of the electron beam. These vacuum considerations necessitate sometimes extensive sample preparation procedures which, in the case of forensic applications are undesirable because they alter the condition of the sample and can raise questions about the value of the analysis as evidence. ESEM and other low vacuum technologies partially isolate the sample chamber from the column with pressure limiting apertures to allow low vacuum conditions in the sample environment. Most samples can be examined without alteration.

The imaging and analytical results generated by forensic investigations must include validation procedures sufficient to meet the legal and ethical standards of their ultimate use, which may include a role in decisions that affect the health, well being, life, or death of a victim or suspect. The instrument and all techniques must be carefully calibrated using traceable standards and reproducible procedures. Automation of the validation procedures not only enhances their reproducibility but also encourages their more frequent use.

A final consideration for the individual forensic laboratory must be the relative costs and benefits of in-house capability compared to outsourcing through a service laboratory. Advanced SEMs and dual beam systems range in cost from less than $150,000 to more than $1,000,000.Clearly, the acquisition requires careful consideration and extensive justification. At some level, the benefits of in-house capability—careful control of the chain of possession of evidence and analytical procedures, faster analytical turnaround, and the ability to do a thorough analysis that adapts and responds to intermediate results—justify the cost.