**3. Application of AFM in forensic science**

This section recapitulates a number of AFM studies that illustrates applicability of AFM in relation forensic traces evidence analysis and its potential for crime investigations or reconstruction.

#### **3.1 Determination of the age bloodstains**

Blood stains are the most common type of forensic evidence found on the crime scene. The blood stains play very important role in the time determination of the actual criminal activity, hence determination of the age of bloodstains can prove to be highly effective in solving the crimes in shorter time duration. This information can provide a good perceptions regarding the victim time of death or to create a link between the suspects to the crime scene at the time when crime was committed. These area has attracted much attention worldwide over the years of various researchers since very few techniques such as electron paramagnetic resonance, high-performance liquid chromatography, quantification of RNA degradation and hyperspectral imaging [20–22]. In the review published by Bremmer et al. [23] they mention about the invasive techniques such as HPLC method, RNA analysis and EPR. Hyperspectral imaging are applied for the same problem. Even though HSI is a promising technology it has high error rate of about 2.7 days as per Edelman et al. [22].

#### *Atomic Force Microscope in Forensic Examination DOI: http://dx.doi.org/10.5772/intechopen.104704*

Research has been done where the application of AFM is explored to study morphological changes of red blood cells (RBCs) to determine the relation with the time of death of individual. Wu et al. [24] has studied, the time-dependent, morphological changes of RBC in three different conditions such as room-temperature condition (controlled), outdoor environmental condition (uncontrolled) and low-temperature condition (controlled) using AFM on clean glass or newly peeled mica. They found that the substrate types have different effects on cellular morphology of RBC. Further, the RBC showed typical biconcave shape on mica and biconcave shape or flattened shape on glass, also the mean volume of RBCs on mica was significantly larger than that of cells on glass. In relation to the time, the changes in cell volume and adhesive force of RBC under the controlled room-temperature condition were similar to those under the uncontrolled outdoor-environmental condition as the time lapse. However, under the controlled low-temperature condition, the changes in cell volume happened mainly due to the RBCs collapse and the adhesive force curves exhibited the high alternations in RBC viscoelasticity. They concluded that AFM has significant application in forensic medicine or investigations, in relation to the estimation of age of bloodstain. **Figure 2** shows the morphological comparison of RBCs on mica (a) and glass.

Chen and Cai did study on the morphological changes in a whole erythrocyte and of the erythrocyte membrane surface ultrastructure using tapping mode atomic force microscopy (TM-AFM) on mica substrate exposed in air over a 5-day period. They observed that the erythrocyte showed deformation of whole cell and membrane surface of unfixed erythrocytes as the time lapse. After 0.5 days of exposure, the fissures and cell shrinkage was observed and at 2.5 days of exposure, the development

#### **Figure 2.**

*Morphological comparison of RBCs on mica (a) and glass (c) and (d). (a) and (d) RBCs in typical biconcave shape. (c) A flattened RBC. (a1) presents a height profile extracted from the cross section indicated by the dashed line in (a). The AFM-measured concave depth (CD) and width (CW) (FWHM) of RBC are 368.2 nm and 3.125 mm, respectively. (c1) and (d1) present the height profiles from the dashed lines in (c) and (d), respectively. The CD and CWin (d) are 219.6 nm and 2.561 mm, respectively. (b) and (e) present histograms of CD (b) and CW (e) of RBCs on various substrates. (f) and (g) indicate the differences of cell volume (f) and adhesive force (g) between RBCs on mica and on glass.*

of nanometer-scale protuberances was observed, also the protuberances number increases with increasing time. Hence the present study presented the application that the changes of cell shape and cell membrane surface ultrastructure can prove to be helpful to estimate the time of death [25].

Lamzin and Khayrullin in their work studied the changes of RBC membranes stiffness in sRBC and the form and size of RBC probed using AFM by storing samples for 35 days at standard temperature conditions as shown in **Figure 3**. Their research revealed that statistically significant increase of YM values of RBC were observed as well as alteration of their form to echinocytes and spheroechinocytes of sRBC within 35 days at +4°C was noticed. They mentioned that this work can prove to be useful as an immediate criteria for applicability of sRBC for blood transfusion [26]. Marco Girasole et al. has exploited the full potential of atomic force microscopy (AFM) to investigate various characteristic of the erythrocytes' life, death and interaction with the environment. As per Marco Girasole et al. [27] AFM is still a continuously growing technique which can be applied for studying more variant information in relation to the RBCs biochemical or biophysical status at different environmental conditions.

Threes Smijs et al. applied atomic force microscopy to investigate the elasticity of RBCs from the peripheral zone of 4–8 day old bloodstains. They observed that the elasticity of six RBCs from a 5 day old bloodstain seemed homogenous with a mean Young's modulus of 1.6 ± 0.4 GPa. As the time lapse, a significant age effect was observed in RBC elasticity that is on 4 days: 0.8 ± 0.1 GPa; 5 days: 1.7 ± 0.9 GPa; 6 days: 2.3 ± 0.6 Gpa; 7 days: 4.5 ± 0.6 GPa; 8 days: 6.0 ± 1.8 GPa; probe spring constants 25.16–67.48 N/m. They found that a bloodstain age determination with a 24 h precision only for 6–7 day old stains can be done. The silicon tip condition was regularly checked using scanning electron microscopy as an increase in bluntness was noticed after four to six cell indentations [28].

Cavalcanti and Silva studied biophysical properties that is morphology and elasticity of RBCs using atomic force microscopy. They aimed to investigate the time since death (TSD) from blood smears by analyzing changes occurring in the RBCs of a group of voluntary. Further, they also investigated that whether any difference in TSD analysis occurs on three different surfaces such as glass, metal, or ceramic after

**Figure 3.** *The AFM image of the dry specimen prepared from sRBC after 1 day (a) and 35 days (b) of storage.*

*Atomic Force Microscope in Forensic Examination DOI: http://dx.doi.org/10.5772/intechopen.104704*

blood smears deposition occurs on these surfaces. They calculated force × distance curves obtained from RBCs membrane surface deformation as a function of time. They observed that there is no appreciable difference in the structure of RBCs over 28 days but significant differences were noticed on glass, metal, or ceramic surfaces. They concluded that the use of AFM in crime scenes still requires the development for accurate estimation of the TSD for blood spots [29]. Strasser et al. also explored erythrocytes in a blood sample to study elasticity changes in a fresh blood spot on a glass slide. At first they found presence of several RBCs in "doughnut-like" structure, which could easily be detected due to their typical "doughnut-like" appearance further the elasticity pattern showed a decrease over time which may be due to alteration of the blood spot during the drying and coagulation process. They concluded that these preliminary data can demonstrates the capacity for development of calibration curves, which have potential in estimation of bloodstain ages during forensic investigations [30]. Different body fluids are also been utilized for the extraction of DNA because of its use as a forensic tool during investigation. AFM can add in the characterization of the "trace DNA" deposited on various surface during any mutual contact. The stiffness of DNA's double strand can be discriminated from its single strand and counting of the copied DNA can be done by using AFM [31].

#### **3.2 Document forgery**

Document examination involves techniques which causes less or no damage to the documents and allows maximum retrieval of data from it. The determination of the sequence of strokes is still a big problem in the field of forensic document examination. Till today the optical microscope are used with different illumination methods and magnifications in determination of sequence of strokes. But the use of same does not provide a reliable results in every cases because of the interaction of the light with crossing ink lines, the depth of focus, low resolving power as well as low magnification range of the optical microscopes. Kasas et al. [32] studied line crossing problem on paper printed form dot matrix printers and different ball-point pens on plain paper. They found that AFM produces qualitatively similar results and overcomes some of the scanning electron microscope limitations, i.e., vacuum and specimen's conductive coating. **Figure 4** shows the cut-outs of crossings of ball-point pen strokes on dot matrix printed letters in newer printer ribbon and worn printer ribbon. They concluded that AFM is a powerful alternative to the SEM for line crossing problem. Brandao et al. in their work has focused on the problem of counterfeiting which involves making an imitation or copy manufactured without the legal sanction of the government. They explored AFM and Raman techniques for the examination of both authentic and counterfeit Brazilian driver licenses, and national and international banknotes. The AFM results showed that the parameters, such as roughness and topographic profiles of the chalcographic region of banknotes and Brazilian driver licenses, can be successfully visually discriminate between authentic and counterfeit documents. They also showed the application of statistical analysis using the Student's t-test which showed that the asymmetry values obtained from series numbers and micro-letter regions can help in identifying the counterfeiting. They also indicated that the paper used to counterfeit the Brazilian driver license and the real banknote was an "office" type with inkjet printing by the use of the AFM technique [33]. Further the combination can also help to recognize the crossing lines between ballpoint pens, and ballpoint pens and printers, to discriminate between genuine and counterfeit medicines, to identify counterfeit documents produced from washing

**Figure 4.**

*Cut-outs of crossings of ball-point pen strokes on dot matrix printed letters. (a) Newer printer ribbon; (b) worn printer ribbon.*

methods, to determine the microstructural information on textile fibers (discriminate between carpets, clothes, cars, etc.) in a crime scene investigation. The combination provides fast, very reliable, and reproducible analysis.

Chen et al. in their work highlighted the quality of AFM compared to SEM for forensic forgery investigations in relation to crossing lines. They examined topographic features of four papers namely duplicator, copper printing, glassine and kraft paper on which crossing lines were done with three different types of oil-based pens as shown in **Figure 5**. For all pens they establish similar differences in height profiles analogous to the inks accumulations at the places where the first pen stroke overlay with the edge of second pen stroke. The work do showed the usefulness of AFM imaging to detect crossing lines under the selected test conditions [15]. As per Ellen, AFM imaging technique can provide high potential in forensic document examination especially in cases to study crossing lines and document forgery cases which can further be explored [34]. Although the many research is been done to prove the usefulness of AFM imaging to detect crossing lines but the overall paper surface roughness hampers the detection of erased, partially erased lines or slightly printed ink patterns on the pages. The height profiles of ink streaks on documents differs on the different types of the paper as the absorption differs. These hinder the correct interpretation of the height images. Though if AFM imaging is applied in these types of investigations the confirmation can only be achieved by usage of other instruments such as Raman spectroscopy to convey the final crucial decisive information.
