Introduction

Forensic science is aimed at detecting and analysing evidential material at crime scenes. Collection and careful laboratory analysis of any trace material is an extremely important activity in order to obtain as many pieces of information as possible. Considerable skill is involved in conducting searches at crime scenes. Frequently, particularly where sexual assault has occurred, crime scene investigators encounter mixed biological traces. The ability to isolate different cell populations (i.e. vaginal/sperm cells, epithelial/white cells, white/sperm cells, etc.) is therefore desirable to ensure the successful outcome of an investigation (Murray and Curran, 2005). Laser microdissection techniques have recently been introduced into forensic analysis (Giuffre et al., 2005) and have proved to be very powerful in the isolation of specific cells from complex mixtures of biological material (Giuffre et al., 2004). It has also increased the types of samples that can be collected from crime scenes and usefully analysed (Plate 10.1).

Laser microdissection techniques fall within the field of microgenomics, which has been referred to as 'a quantitative molecular analysis of nucleic acids or proteins obtained from a single cell or a tiny amount of cells, which were isolated, collected and examined according to precise micromanipulating techniques'.

Molecular Forensics. Edited by Ralph Rapley and David Whitehouse Copyright 2007 by John Wiley & Sons, Ltd.

Micro-isolation and micromanipulation techniques are essential when undertaking laser microdissection techniques. Laser capture and laser cutting are the main techniques applied to perform cell micro-isolation.

Emmert-Buck and colleagues (1996) developed the first laser capture microdissection device, which was patented by Arcturus in the following year (Plate 10.2). It was based upon the heating and fusion of a thermo-sensitive plastic polymer that is modified by low-energy laser pulses (with near-infrared wavelength) (Plate 10.3). This is also a useful device when dealing with RNA because of its sensitivity to high temperatures. However, the drawback is that the method lacks sufficient precision to isolate target cells from the matrix (Plate 10.4).

Laser cutting is preferable because the precision of the laser allows the isolation of single cells or a group of cells from more complex biological matrices such as tissue sections or hair shafts. Table 10.1 compares the main features of laser cutting and laser capture microdissection techniques.

Modern laser cutting devices exploit a solid-state UV laser beam with a wavelength between 337 nm and 370 nm. Such a beam is able to behave like an electromagnetic knife and can destroy whatever it meets, including the biological substrate where cells are usually found. The cutting is done with great precision down to a single micrometre.

The cell sample is focused with the aid of a microscope through which the operator can select the target and then cut using the laser beam. The three main laser cutting systems may be defined by the collection method used:

• Collection by gravity.

• Collection by an adhesive polymer applied on a tube cap.

• Collection through catapulting or energetic pulse.

Laser microdissecting instruments are coupled to different microscope systems depending on the collection method to be used. For example, a conventional upright microscope may be used for gravity-based collection, while an inverted microscope is used for the other two systems of collection.

Table 10.1 Comparison of laser capture microdissection and laser cutting techniques

Laser capture microdissection

Laser cutting

Infrared beam

Ultravidet beam

Suitable for isolation of a small number of

Suitable for wide sections

cells

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