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Who Do We Think We Are?

Blue, brown, or green eyes? Which hair color? Which illnesses may affect us? Whose child am I? All of this information and much more is stored, or at least set, in our genes. In crime novels, but also in real life, genetic analysis is called upon for advice when it comes to finding an answer to the question, "Who did it?". The "genetic fingerprint" has become an often consulted and unmistakable evidence. Last but not least, genome analysis holds the key to groundbreaking discoveries for many health-related questions.

The crucial basis for it, is the possibility of a holistic analysis of genomes. This holistic analysis is therefore becoming increasingly important. A leading technique is the "sequencing-by-synthesis" method, whose principle is based on fluorescent microscopy. Since its development, this method has made enormous progress. Speed and precision have increased by several orders of magnitude, while costs have also decreased by several orders of magnitude. The positioning techniques employed in the microscope systems used for this purpose have also contributed significantly to the progress.

DNA Recombination Made Visible by Detecting Fluorescence Signals

In the "sequencing-by-synthesis" method, so called "flow cells" of nucleotide with fluorescent markers are gradually incorporated into a single strand DNA (nucleic acid chain) and are, as such, 'synthesized'. If it comes to a recombination of the base pair, a characteristic fluorescent color signal is generated in the visual spectrum for each of the four different base pair combinations. The four colors are: Blue, green, yellow, and red. These signals can be detected microscopically.

Since the signals are weak and also very short lived, the detection and imaging process must be very fast. For this purpose, two coordinated positioning processes are necessary.

First of all, the flow cell must be precisely positioned under the objective. For this, PI offers high-precision XY stages. Depending on the size of the flow cell and its actual geometric shape, the flow cell gets analyzed over a travel range of more than 100 mm in size – this must be done quickly but with high precision in order to detect all signals.

In addition, the objective must be precisely aligned to the occurrence along the optical axis, within the depth of field of the lens. Depending on the magnification factor of the lens, the depth of field may be in the range of a few hundred nanometers. Not only is the relevant precision key, but also speed and dynamics. Because, on the one hand, the exact level of the occurrence (fluorescence signal) cannot be predicted and, on the other hand, possible tolerances of the flow cell as well as slightest errors due to pitch/yaw of the the XY positioning stages must be levelled out by the motion.

For Z focusing, PI offers both PIFOC® objective positioning stages, which are based on lever-amplified piezo actuators, and, alternatively, highly dynamic voice coil drives with large travel ranges.

 

For more details on PI products for genome analysis, see the "Genome Sequencing - Sequencing-by-Synthesis Method" brochure.

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